Starch mobilization robustly paces to dawn despite severe disruption of the transcriptional circadian oscillator. Arabidopsis thaliana), accumulate starch in the daytime and remobilize it to support maintenance and growth at night. Starch accumulation is increased when carbon is in short supply, for example, in short photoperiods. Mobilization is paced to exhaust starch around dawn, as anticipated by the circadian clock. This diel pattern of turnover is largely robust against loss of day, dawn, dusk, or evening clock components. Here, we investigated diel starch turnover in the triple circadian clock mutant lhy cca1 elf3, which lacks the LATE ELONGATED HYPOCOTYL and the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) dawn components and the EARLY FLOWERING3 (ELF3) evening components of the circadian clock. The diel oscillations of transcripts for the remaining clock components and related genes like REVEILLE and PHYTOCHROME-INTERACING FACTOR family members exhibited attenuated amplitudes and altered peak time, weakened dawn dominance, and decreased robustness against changes in the external light–dark cycle. The triple mutant was unable to increase starch accumulation in short photoperiods. However, it was still able to pace starch mobilization to around dawn in different photoperiods and growth irradiances and to around 24 h after the previous dawn in T17 and T28 cycles. The triple mutant was able to slow down starch mobilization after a sudden low-light day or a sudden early dusk, although in the latter case it did not fully compensate for the lengthened night. Overall, there was a slight trend to less linear mobilization of starch. Thus, starch mobilization can be paced rather robustly to dawn despite a major disruption of the transcriptional clock. It is proposed that temporal information can be delivered from clock components or a semi-autonomous oscillator.Many plants, including Arabidopsis ( Arabidopsis thaliana), leaf starch is the major transitory C reserve. Diel starch turnover is exquisitely regulated. A larger proportion of the fixed C is accumulated as starch when less C is available over the entire 24-h cycle, for example in short photoperiods or low irradiance (Metabolism and growth in the daytime are driven by photosynthetic carbon (C) fixation but at night depend on reserves accumulated in previous light periods . In manyradiance . This isradiance . At nighradiance . This paradiance .circa diem) regulation. Many clock mutants show a modified pattern of starch mobilization, with starch being exhausted before dawn in lhy cca1, and incompletely exhausted at dawn in PSEUDO-RESPONSE REGULATOR (prr7 prr9), elf3, and GIGANTEA , followed by day , dusk , and evening  genes, which repress outputs like the PHYTCHROME-INTERACTING FACTORS (PIFs) 4 and 5 and the day genes. Later in the cycle, dusk and evening genes decay or self-repress, and LHY and CCA1 expression rises to peak around the next dawn. In addition, three members of the REVEILLE (RVE) family, RVE4, RVE6, and RVE8 positively regulate clock components, especially dusk and evening genes such as TOC1 and LUX  and time to dawn (T) is integrated to set the rate of starch mobilization (R) such that starch is exhausted at dawn  in that mutant (see above). Crucially, mutants lacking dawn, day, dusk, or evening clock components are still able to slow down mobilization after a sudden early dusk  were grown in a 12-h photoperiod at 160 µmol m−2 s−1 . The response  or small  changes in their timing. Summarizing, there is a far larger disruption of the transcriptional circadian clock in lhy cca1 elf3 than in mutants previously investigated for diel starch turnover.We compared the transcript dynamics in  parents , central parents . Some felhy cca1 elf3 in a 12-h photoperiod might be a default response in a nonchallenging condition, or might reflect an unexpected robustness of the network that delivers the temporal input. We therefore investigated diel starch turnover in Ws-2 and lhy cca1 elf3 growing at lower irradiance or in a shorter photoperiod, as well as in non-T24 cycles. We also asked if lhy cca1 elf3 can adjust starch mobilization to a sudden change in irradiance or the timing of dusk  do not overlap between photoperiods), the response was much weaker in lhy cca1 elf3 .As previously reported (see “Introduction”) Ws-2 accumulated starch more rapidly in short photoperiods than in long photoperiods  and alwaoperiods . The ratoperiods . Whereaslhy cca1 elf3 in lower irradiance or a shorter photoperiod led to a slowing down of starch mobilization .Comparison of the temporal kinetics of starch mobilization in Ws-2 and  content . The rel content . The rel−2 s−1 irradiance in a 12-h photoperiod, and 160 and 90 µmol m−2 s−1 irradiance in the 6-h photoperiod were 24.4, 24.2, 23.7, 24.5, and 25.2 h, respectively. The corresponding values in the triple mutant were 25.8, 25.6, 25.3, 23.5, and 24.3 h. Thus, compared to wild-type plants, in the triple mutant the estimated time of StEx is slightly delayed in a 12-h photoperiod (by ∼1.5 h) and slightly advanced in a 6-h photoperiod (by ∼1 h). Incidentally, the values for Ws-2 in this study resemble earlier studies with Col-0 or Ws-2 growing in a 12-h photoperiod at 160 µmol m−2 s−1 irradiance  with starch being exhausted before dawn in lhy cca1 and being incompletely mobilized at dawn in elf3  cycle, or a 14-h light/14-h dark (T28) cycle , wild-type plants set a rate of mobilization that does not completely exhaust starch at dawn in a T17 cycle, and that exhausts starch before dawn in a T28 cycle . Whilst 28 cycle . However28 cycle .lhy cca1 and elf3. As previously reported, starch was exhausted before dawn in lhy cca1 and incompletely mobilized at dawn in elf3 in a T24 cycle  and slower starch mobilization in elf3.For comparison, we investigated starch turnover in different T-cycles in 24 cycle , centralype Ws-2  confirmelhy cca1 elf3 and wild-type Ws-2 and published data . To allow comparison of the kinetics of mobilization across all genotypes and T-cycles, we restricted our analysis to a time range that corresponded to the duration of the night in a T17 cycle (i.e. dusk and the first 8.5 h of darkness). This time range, which corresponded to the first 71% and 61% of the night a T24 and T28 cycles, respectively, provided a good estimate of the initial rate of mobilization set after dusk. Multiple nested linear fits on starch content in this time range were extrapolated to zero starch content to estimate the apparent StEx . app .In wild-type plants, projected values of StExI limits . Thus, iapp between the T17 cycle and the T28 cycle with the 11-h shift in the duration of the external light–dark cycle. StExapp increases by 3.8 h in wild-type Ws-2 and 3.6 h in lhy cca1 elf3, compared to 5.8 h in lhy cca1 and 5.1 h in elf3.The stringency of the 24-h rhythmicity of starch turnover can also be gaged by comparing the magnitude of the increase in StExapp values projected by multiple nested estimations resembled StEx values obtained by regression across all time points in the night . There was good agreement for Ws-2 under different irradiance in a 12-h light/12-h dark cycle and a 6-h light/18-h dark .We checked that the StEx compare . There w compare , but prolhy cca1 elf3 maintains a near-24-h rhythmicity for starch turnover in non-T24 cycles. It maintains 24-h rhythmicity as robustly as wild-type Ws-2 and more robustly than its parents.Together, these analyses show that lhy cca1 elf3 is able to adjust the rate of starch mobilization after a sudden change in conditions. To do this we grew lhy cca1 elf3 in a 12-h photoperiod at 160 µmol m−2 s−1 and then subjected it, on the harvest day, to lower irradiance for one light period or to a sudden early dusk (see We next asked if −2 s−1), reducing irradiance to 90 µmol m−2 s−1 for one day resulted in a 42% decrease in dusk starch content in Ws-2 and a slightly larger 52% decrease in lhy cca1 elf3  or time points where starch content was below that at the time at which the treated plants were suddenly darkened (ZT16–ZT24). The initial rate in the early dusk treatment was calculated excluding time points when starch was lower than at dawn in the control treatment (i.e. excluding time points after about ZT18). In the four experiments, the estimated decrease in mobilization rate was ∼15% and 12%  but not DIN6 transcript was lower in lhy cca1 elf3 than wild-type Ws-2. DIN1 and DIN6 transcript rose in the first 2 h of the night, reaching similar values in both genotypes. This may reflect transient C depletion, because there is a short delay until starch degradation starts after sudden darkening  transcript levels were low throughout the 24-h cycle in wild-type Ws-2, but rose in the second part of the night in lhy cca1 elf3. These results indicate that C-starvation signaling is more strongly activated at night in lhy cca1 elf3 than wild-type Ws-2, in agreement with the slightly lower sugar levels at night in lhy cca1 elf3 than Ws-2.We also investigated the responses of three C-starvation reporter genes in a standard 12-h photoperiod . In the lhy cca1 elf3 has similar or higher levels of sugars than Ws-2 in the light, indicating that its low rate of starch accumulation in short photoperiods is not an indirect effect due to allocation of more C to growth. They also show lhy cca1 elf3 paces mobilization to dawn even though its night-time sugar levels are often lower and C starvation reporter transcripts are higher than in wild-type Ws-2. Overall, diel changes of sugar levels are larger in lhy cca1 elf3 than wild-type Ws-2 indicating a slightly weakened ability of the triple mutant to maintain sugar homeostasis.Taken together, these analyses show that lhy cca1 elf3 is grown in a recurring 12-h photoperiod under relatively high irradiance. We investigated the residual clock in other conditions when less C is available or when the plant is subjected to sudden perturbations  and later in an 18-h photoperiod . RVE4 transcript in Ws-2 and lhy cca1 elf3 peaked at ZT20.8 and ZT21.8 in a 6-h photoperiod, respectively, and only slightly later at ZT22.0 and ZT22.4, respectively, in an 18-h photoperiod.Diel oscillations of  to Ws-2 . RVE4 anPIF transcripts were strongly damped in lhy cca1 elf3, due mainly to abundance remaining high throughout the cycle , PRR9, five RVE, and two PIF family members, and GBSS1 transcript could be scored in lhy cca1 elf3. None of them differentiated between dawn and dusk alignment. We also performed two-way ANOVA to test whether the 6- and 18-h photoperiod time series were significantly different (i.e. desynchronized) when they were aligned to either dawn or dusk  advance in the response of most transcripts in the following night. In lignment . This anlhy cca1 elf3 is still paced by an internal 24-h rhythmicity . Nevertheless, elf3 was able to speed up starch accumulation in short photoperiods . These conclusions were based on a largely qualitative analysis. Whilst assessing if mobilization is modified in ype Ws-2 .These analyzes revealed that starch mobilization is paced rather precisely to 24 h after the previous dawn in a T24 cycle  might gain by explicit consideration of this nonlinearity, which presumably reflects the precision of the underlying regulatory network. It might also be relevant to consider whether the network that regulates the rate of mobilization acts not only to slow down mobilization early in the night when starch content is high, but also to maintain rates of mobilization later in the night when starch content is low.lhy cca1 elf3 retained the ability to pace starch mobilization to close to dawn. It achieved this across a wide range of photoperiod and irradiance growth regimes  but not in a T28 cycle (ZT24.6). This might indicate that the clock imposes its rhythmicity on starch mobilization when the duration of the external light–dark cycle is close to the endogenous rhythm but not when the external cycle differs strongly from the internal period. An analogous argument can be made for the elf3 mutant. Clock period cannot be scored because elf3 is arrhythmic in free-running conditions, but in light–dark cycles elf3 does show delayed peak times of dawn transcripts compared to wild-type plants .Strikingly, starch mobilization was more resilient against the external T-cycle in the triple mutant than in its parents. lhy cca1 elf3 paces starch mobilization to ∼24 h after the previous dawn. One is that regulation of translation, protein stability, or posttranslational modification maintains dawn-entrained oscillations at the level of core clock proteins abundance or functionality, and that these regulate starch mobilization. This explanation seems unlikely, as it would require that the protein or proteins are able to generate an output that regulates starch mobilization, but cannot correctly regulate canonical targets within the clock oscillator itself. It is in principle possible that gating of an output from a nonoscillating clock component might impact rhythmicity to starch turnover. However, this would require that the postulated gating mechanism is robust against the severe disturbance of the clock in lhy cca1 elf3.Several explanations can be offered for how PRR9 transcript did show oscillations in the triple mutant, these were strongly modified from those in wild-type Ws-2 and showed shifting alignment to dawn across different photoperiods, and poor 24-h rhythmicity in in non-T24 cycles. ELF4 transcript retains an oscillation but with strongly altered phase, poor 24-h rhythmicity in non-T24 cycles and is anyway unlikely to be functional in the absence of ELF3 protein. One possible set of candidates might be some members of the RVE family. RVE4, RVE6, and RVE8 are positive effectors of clock genes especially dusk and evening genes such as TOC1 and LUX  in T24 cycles . It is noteworthy that lhy cca1 elf3 exhibits slightly larger diel changes in sugar levels and in the abundance of C-regulated transcripts. This raises the question whether signals deriving from metabolism compensate for a severe attenuation of clock function and maintain the near-wild-type pattern of starch mobilization that is found in the triple mutant. Irrespective of details, it can be envisaged that diel starch turnover is regulated by a flexible multi-layered network with partly redundant components. This complexity may be required to allow plants to, on the one hand, robustly pace starch degradation to dawn across a wide range of growth conditions, and, on the other hand, rapidly adjust the rate of starch degradation in response to day-to-day fluctuations in conditions.In conclusion, the stimulation of starch accumulation in short compared to long photoperiods is attenuated in the higher-order circadian mutant A.thaliana) wild-type accession Ws-2, the lhy cca1 elf3 triple mutant  mutation results in a premature stop codon after the fortieth amino acid, as described by lhy-21 cca1-11 double mutant (Ws-2 background) is the result of a crossing between two T-DNA insertion lines, lhy-11 and cca1-11 described by lhy-21 cca1-11 elf3-4 triple mutant was obtained by crossing the lhy-21 cca1-11 double mutant and the elf3-4 single mutant . Irradiance was decreased by dimming of tubular fluorescent lamps. Data for stable 24 h cycles was compiled from three sets of experiments: (1) lhy cca1 elf3 in 12/12-h light–dark cycle at 160 µmol m−2 s−1  lhy cca1 elf3 and Ws-2 in 6/18-h light–dark cycles at 160 µmol and 90 m−2 s−1. Comparisons of results in stable non-24 h were done using independent experiments conducted in 17- and 28-h diel cycles in combination with results from the above set of experiments (1) in 24 h diel cycles. Experiments in fluctuating conditions were always conducted in parallel with a control group (i.e. sudden early dusk and sudden low light). The protocol on the harvest day is described in Seeds were sown (spread in excess) on wet soil  mixed with vermiculite using plastic pots (10 cm of diameter) in 30 × 50 × 6 cm plastic trays. To avoid different developmental stages due to differing germination speed between genotypes, pots were initially kept for 1–3 days in a cold room (4°C) before transfer to growth chambers . Pots were covered with transparent plastic lids during the 5 initial days after sowing (DAS). Standard parameters for growth are provided in lhy cca1 elf3 mutant and Ws-2 wild-type plants using ZT0 samples of the 17, 24, and 28 h diurnal cycle experiments indicated in Fresh weight per plant was estimated for both RNA was extracted and transcripts analyzed by RT-qPCR . Absolute quantification of copy number per gram of fresh weight was performed using artificial RNA internal standards as in Starch and metabolites were assayed  as previously .All data analyses and their visualization were performed using the open source R language and its publicly available packages .Significance of changes in metabolite and transcript levels was tested using analysis of variance ANOVA; , compariTime of peak was calculated based on the average peak timing of bootstrapped time series using inflexion points in both polynomial fit and spline smooth (50 repetitions for each method). Fits were optimized for minimum second-order Akaike’s Information Criterion. Peak amplitude of circadian oscillation was defined as the difference between maximum and minimum mean expression level for a given gene in a given condition. Gaussian error propagation  was usedt distribution using the standard error of the slope.Rates of starch accumulation and mobilization were estimated as the slope of the fit linear models describing starch levels as a function of time, applied to data  for the light and dark periods, respectively. Rates of starch mobilization in the sudden early dusk experiments were calculated using smaller subsets of the dark periods. The 95% CI was estimated from a normal Student’s app was estimated by extrapolating the initial rate of starch mobilization, using multiple fittings to a progressively larger number of consecutive time points  using starch content at dusk and in the first 8.5 h of the night. Final results were averaged  and reported in combination with its 95% CI .In experiments with different growth photoperiod and irradiance in T24 cycles, the expected time of StEx was estimated from the root of the linear fitting  = 0) using a nonderivative approach . In non-A straight line was interpolated between dusk and dawn starch reference values to provide, at any given time, a predicted starch content if starch mobilization were to be linear. At each harvest time point, the predicted starch content was subtracted from the measured starch content, and the difference (residue) normalized on the predicted starch content. These normed residues were then averaged across all time points. The 95% CI of the nonlinearity score was calculated using a nonparametric bootstrap procedure . The scoAccession number/identifier of genes cited in this article can be found in The following materials are available in the online version of this article. Experimental design. Biomass and chlorophyll. Diel changes of clock transcript abundance in a stable 12–h light/12–h dark cycle at 160 µmol m−2 s−1 irradiance. Diel starch turnover in wild-type Ws-2 and the triple lhy cca1 elf3 mutant, replotted with starch content normalized on the dusk starch content. Analysis of the dynamics of diel starch turnover in wild-type Ws-2, lhy cca1 elf3 and its parents lhy cca1 and elf3 in different T-cycles. Response of starch degradation after a single day of low irradiance, with starch normalized on dusk starch content. Response of starch mobilization after a sudden early dusk, three further experiments that replicate the experiment of  Sugar content. Diel response of transcript abundance in different photoperiods. Two-way ANOVA test of whether the oscillations of transcript abundance in different photoperiods and after a sudden early dusk are dawn-or dusk-aligned in Ws-2 and lhy cca1 elf3. Response of transcript abundance to a sudden early dusk. Response of transcript abundance in different T cycles. Response of transcript abundance in an extended night. Dynamics of transcripts after a sudden early dusk and in non T24 cycles, and acute response of clock transcripts to light and dark. TAIR, UniPROT, and EMBL accession number/identification. Metabolite content and biomass accumulation. Transcript abundance. Estimated peak time and amplitude of the diel oscillations of clock and RVE4, RVE8 and GBSS1 transcript abundance in different photoperiods. Mutual Information analysis of the photoperiod and early dawn time series. Two-way ANOVA of the photoperiod and sudden early dusk time series.kiac226_Supplementary_DataClick here for additional data file.


Arteriovenous graft (AVG) failure continues to be a life-threatening problem in haemodialysis. Graft failure can occur if the implanted graft is not well-matched to the vasculature of the patient. Likewise, stenosis often develops at the vein-graft anastomosis, contributing to thrombosis and early graft failure. To address this clinical need, a novel ink formulation comprised of ACMO/TMPTA/TMETA for 3D printing a AVG was developed (ACMO-AVG), in which the printed AVG was biocompatible and did not induce cytotoxicity. The ease of customizing the ACMO-AVG according to different requirements was demonstrated. Furthermore, the AVG displayed similar mechanical properties to the commercially available arteriovenous ePTFE graft (ePTFE-AVG). Unlike ePTFE-AVG, the ACMO-AVG displayed excellent anti-fouling characteristics because no plasma protein adsorption and platelet adhesion were detected on the luminal surfaces after 2 h of incubation. Similarly, exposure to human endothelial cells and human vascular smooth muscle cells did not result in any cell detection on the surfaces of the ACMO-AVG. Thus, the present study demonstrates a newly developed 3D printing ink formulation that can be successfully 3D printed into a clinically applicable vascular access used for haemodialysis. An arteriovenous graft that was successfully 3D printed with a novel printing ink formulation that displayed excellent mechanical and anti-fouling properties. There are two different dialysis methods, peritoneal dialysis and haemodialysis, with the latter most commonly applied to ESRD patients. The majority of ESRD patients receive haemodialysis two to three times a week.2 Patients that require haemodialysis are implanted with either an arteriovenous fistula (AVF) or an arteriovenous graft (AVG) under the skin that bridges an artery and a vein, providing needle placement access for haemodialysis.3 Clinically, the AVF is the preferred method when compared to AVG since the former method has a relatively low risk of long-term complications and better long-term patency. However, AVF placements are susceptible to early failure and have a 20–60% chance of not providing enough flow enhancement to facilitate sufficient dialysis. It has been estimated that the early failure rate of AVF placements is 33.8%.4Globally, the number of patients with end-stage renal disease (ESRD) continues to rise.5 This is a drawback for AVG placements since a significant number of ESRD patients only have smaller vessels available for graft implantation. Grafts with unsuitable diameters can cause vascular steal in the adjacent vessels, which can lead to stenosis.6 Furthermore, immediate stenosis may happen if twisting and kinking occurs during the graft's placement. If the graft needs to be placed in a looped configuration, tunnelling the loop too tight can lead to kinking at the apex of the graft.7 Therefore, it is ideal if the design and fabrication of the graft can be customized according to the vasculature of each individual patient, preventing the occurrence of twisting and kinking.Maturation failure is the most common reason that contributes to the early failure of AVFs, in which patients will need to switch to AVGs until a suitable AVF is available. Unlike AVFs, AVGs enable quicker vascular access in haemodialysis patients since cannulation can start within two to four weeks after placement as opposed to months with AVF placements. Synthetic AVGs such as expanded polytetrafluoroethylene (ePTFE) have been clinically used for decades, but can only be applied to vessels with large diameters (≥6 mm).8 IH not only results in haemodialysis failure, it can potentially cause mortality. Although attempts have been made to modify the surfaces of ePTFE through the addition of heparin9 or nitinol,10 the clinical results indicate that these modifications can only prevent the development of IH at an early stage after the initial graft placement. It has been estimated that most haemodialysis patients require graft replacement 12 months after the initial graft implantation.11 Since a significant number of haemodialysis patients are >65 years old, constant graft replacement creates a significant toll on their health and increases the chance of bacterial infection. Thus, in addition to a fabrication method that allows physicians to custom-design AVGs with minimum technical skill, there is an urgent need to develop a novel material with properties that minimize the chance of developing IH.In addition to improper placement, the development of intimal hyperplasia (IH) is another major cause of AVG failure. Studies have shown the graft function of ePTFE vascular grafts in ESRD patients at 12 months after surgery varies from 10% to 78% because of IH.12 Similarly, human acellular vessels have also been demonstrated to be viable option to replace the commercial arteriovenous ePTFE graft (ePTFE-AVG).13 However, either method will require an experienced technician and expensive equipment. It is believed 3D printing is the future for fabricating custom-designed AVGs that will meet all the needs of a haemodialysis patient. In the present study, the physical and biological characteristics of a novel designed AVG have been investigated, in which the graft was printed with a novel printing ink formulation using a 3D printer. The physical and mechanical properties of the AVG were analysed, in which the tensile strength and suture retention strength were measured. In addition, the 3D printed graft was examined for its anti-fouling effects against plasma proteins, human platelets and human endothelial and vascular smooth muscle cells.A recent study has demonstrated electrospinning as an effective method to prepare custom-designed AVGs.14 Already, there have been numerous studies demonstrating the potential of 3D printed acrylic polymers for different biomedical applications.15 Since the 3D printed AVG needs to have viscoelastic and anti-fouling properties comparable to the ePTFE-AVG, the different acrylic polymers that can be 3D printed were evaluated. It was decided that two different 3D printing ink formulations would be examined for their applications as vascular access, 4-acryloylmorpholine (ACMO) and PEGDA (PEGDA575/250)  technology, in which the printed materials are solidified when exposed to a near UV light source. In particular, acrylic polymers are the materials commonly used as the printing materials for DLP due to their rapid curing reactions and high resolution optical sources, in which the materials become solidified through the establishment of acrylic bonds after being exposed to UV-light.) Fig. S1. Polymer22 It was noticed that the structural integrity of the printed AVG was greatly influenced by the design of the outer rim  was structurally stable after being 3D printed with twisted outer rim  software, different designs of the arteriovenous ACMO graft (ACMO-AVG) were proposed . Just liuter rim . Unlike uter rim . Since c Fig. S2a.24 which can occur when one layer fails to bond correctly with the upper or lower layer. Apart from an impropriate design, factors such as the polymer composition, room temperature and printing speed can also contribute to layer splitting.25 Since it is desirable to have an AVG ready within the shortest possible timeframe, the speed setting of the 3D printing protocol allowed a 20 cm ACMO-AVG to be prepared in 12 h. It is possible to 3D print the ACMO-AVG with a cylindrical outer rim without layer splitting, although the fabrication time would be significantly increased. Nevertheless, it was clear that the ACMO-AVG with the twisted outer rim design was structurally more stable than the other designs and that it had less signs of layer splitting when compared to PEGDA-AVG. Consequently, the ACMO-AVG was the main focus for the subsequent experiments.To further understand how the twisted outer rim maintained the structural integrity of the ACMO-AVG, the surface structure of the graft  was exam26 The customizability of the 3D printed ACMO-AVG was demonstrated by showing the diameter of the graft could be easily adjusted to either 4 or 6 mm .A sample . Consequ31 Similar results were also obtained by calculating the fracture energy of the three AVGs  for 72 h at 37 °C. Subsequently, the pre-exposed media was removed from the 3D printed material and used to culture WS1 for another 72 h at 37 °C. When compared to the positive control, which was WS1 cultured in normal media, the cell viability of the cells cultured the different pre-exposed media all showed similar cell viability  for some medical applicationsiability  and S4†.44 Activated platelets release cytokines that recruit immune cells and amplify the coagulation cascade. Consequently, it has become a clinical standard that the luminal surfaces of AVGs need to prevent fouling by the plasma proteins.44 To evaluate the anti-fouling property of ACMO-AVG, the graft was cut normal to the long axis into small strips dimension of 20 mm × 5 mm. The luminal surfaces of the trimmed strips were then exposed to outdated human platelet rich plasma (PRP) at 37 °C for 2 h with shaking. The unbound platelets and plasma proteins were washed off with PBS and the anti-fouling characteristic assessed using anti-human GPIIb and anti-human fibrinogen antibodies  and human vascular smooth muscle cells (hVSMCs) overnight at 37 °C. When compared to the number of HUVECs detected on the cell culture plate, a lower number of cells was detected on the surface of the ePTFE-AVG  and vascular smooth muscle cells (VSMCs) also promote the development of IH in AVGs. The uncontrolled growth of ECs and VSMCs at the graft-vein anastomosis leads to IH development at the connected junction, which accelerates graft failure.PTFE-AVG . Both thPTFE-AVG .49 A study in a porcine model showed that ePTFE-AVG coated with anti-CD34 antibodies, which are antibodies that specifically target endothelial progenitor cells, allowed at least 85% of the graft surface to be covered with ECs within 3 days of implantation.50 However, when compared to the uncoated ePTFE-AVGs, which only had 32% of the graft surfaces covered with ECs, IH at the venous anastomosis was strongly increased in the anti-CD34–coated grafts. This indicated that rapid endothelisation accelerates the development of IH in implanted AVGs rather than preventing or minimizing the pathological process. Likewise, ePTFE-AVG coated with either anti-thrombotic peptides51 or heparin52 did not display any significant improvement in down-regulating IH when compared to the uncoated graft. Therefore, it is clinically advantageous that the core material components of the graft exhibit strong anti-fouling characteristics (e.g. ACMO) rather than depending on an additional coating for its anti-fouling effects.It has been envisaged that IH could be minimized if the implanted graft was rapidly covered with endothelial cells, thus preventing thrombosis and immune recognition.53,54 Contrary to the synthetic AVGs, the re-cellularisation of TEVGs with ECs and VSMCs did not result in the extensive development of IH or an immunogenic response at 6 months after the implantation in large animal models. Moreover, since the TEVGs consist of secreted extracellular matrix (ECM) proteins, the mechanical properties are similar to those in a recent phase II clinical trial; TEVGs developed with the VSMCs derived from deceased organs and tissue donors had a 12 months primary patency of 73%, which was similar to the ePTFE controlled group and did not display any post-cannulation bleeding after dialysis.55 Even though TEVGs seem to be a viable alternatives to ePTFE-AVGs, the cost for making one TEVG is likely to be unbearable for most haemodialysis patients. Extensive genetic screening needs to be performed for each batch of donor cells, and a minimum of 8 weeks is required for pulsatile cyclic distension. Moreover, the customization of TEVGs according to an individual patient's vasculature is likely to be challenging, as opposed to the 3D printed ACMO-AVG reported in the present study.Recently, there have been numerous reports on developing tissue engineered vascular grafts (TEVGs) for haemodialysis.As the number of patients requiring haemodialysis continues to increase annually, the demand for custom-designed AVGs is becoming an important need. The present study demonstrates that a customized AVG can be easily fabricated using a standard desktop 3D printer using a novel 3D printing ink formulation comprised of ACMO/TMPTA/TMETA. The ACMO-AVG was demonstrated to have strong tensile strength and was thermo-stable under physiological conditions. Moreover, the ACMO-AVG was shown to be non-toxic to human cells and displayed anti-fouling characteristics that prevent plasma protein deposition, platelet adhesion and endothelialisation. It is envisioned that in the future every haemodialysis centre could utilize 3D printing technology to manufacture their own AVGs to meet the demands of their ESRD patients.The 3D printing resin formulation used for the arteriovenous ACMO-based graft (ACMO-AVG) was comprised of 4 mL of 4-acryloylmorpholine , 0.5 mL of trimethylolpropane triacrylate  and 0.5 mL of trimethylolpropane ethoxylate triacrylate  in 1 mL of ethanol. Likewise, the 3D printing ink formulation of the arteriovenous PEGDA graft (PEGDA-AVG) was comprised of 60% PEGDA-575, 30% PEGDA-250  and 10% ethanol. Bis-phenylphosphine oxide was used to as the photoinitiator in the printing process. Computer assisted design (CAD) models were first drawn and the different structural features were specified. The models were then sliced into multiple images using the manufacturers provided software. All the 3D printing processes were conducted using a DLP 3D printer. For the purpose of this study, the curing time for both the ACMO-AVG and PEGDA-AVG were kept the same and after the printing process, the grafts were first rinsed with ethanol before being subjected to a post-curing exposure to 365 nm UV light for 2 min to ensure the full extent of polymerization.−2) for 5 min. Subsequently, all the samples were freeze-dried  for 24 h to ensure all the water was removed. Prior to loading the samples into the field emission scanning electron microscope , the samples surfaces were coated with platinum gold (10 mA) for 60 s.The ACMO-based printing ink formulation was 3D printed in the dimensions of 5 mm × 5 mm × 5 mm. The samples were then cleaned with 75% ethanol and further exposed to UV light . All the samples were loaded into textured grips horizontally and pulled from both directions simultaneously at the rate of 0.1 mm s−1, in which the force required to pull the suture through the samples was recorded as the suture retention strength.One end of the 3D printed samples (30 mm × 10 mm × 5 mm) was connected to the textured grip of a dynamic mechanical analyser (Tytron™ 250 Microforce testing system), while the other end had a 5-0 prolene suture passed through and knotted 5 mm from its edge. The suture was pulled at a rate of 50 mm min−3 (milli-joules per milli-meter squared).Fracture energy analyses were determined by integrating the area under the stress–strain curve. The end points for the integration are the points of fracture, which represent the overall toughness of the analysed samples and the energy absorbed by the materials before break. The unit was expressed as mJ mm−1.The 3D printed ACMO-AVG  was connected to the outflow tubing of a circulating water bath and that the other end of the graft was connected to another tubing. Both connections were sealed with parafilm to ensure no water leakage occurred while the water was flowed through the graft. The circulating water bath was set at 37 °C with a flow rate of ∼9.8 L min2 atmosphere. Human vascular smooth muscle cells  were cultured in M199  with 10% foetal bovine serum (FBS) and maintained at 37 °C under a 5% CO2 atmosphere. The conditioned media used for both mammalian cell lines were changed every two days and passaged when the cell confluency reached ∼80%.Batches of outdated human platelet rich plasma (PRP) were purchased from Taiwan blood service foundation , in which any experimental works involved the use of human PRP were approved by the research ethics committee for human subject protection, National Chiao Tung University. Human umbilical endothelial cells  were cultured in endothelial cell medium  and maintained at 37 °C under a 5% COr = 0.8 cm), which could fit into a 24-well plate. Likewise, sections of ePTFE were excised from a commercial ePTFE-AVG into a size that fitted in the well of a 24-well plate. The 24-well plate containing the 3D printed ACMO sample, PEGDA sample and trimmed sections of ePTFE were filled with either DMEM containing 10% FBS or PBS and incubated at 37 °C under a 5% CO2 atmosphere for 72 h. Subsequently, the weight of the three materials were determined and compared to their original weights. To determine if the materials had any toxic effect on the cell viability, the 3D printed ACMO sample, PEGDA sample and trimmed sections of ePTFE were firstly incubated in DMEM containing 10% FBS at 37 °C under a 5% CO2 atmosphere. The media were then extracted and used to culture human skin fibroblast  at 37 °C under a 5% CO2 atmosphere for 72 h. The cell viability was then determined using a hemocytometer to count the number of dead cells versus the number of live cells.Either the ACMO-based or PEDGA-based printing ink formulation was 3D printed into a disc shape (6 platelets per mL were seeded onto PEGDA575/250 (3 : 2), PEGDA575/250 (2 : 3) and a section of ePTFE in a 24-well plate for 2 h with agitation at 37 °C. The plate was then washed with Tyrode's buffer  and the bound PRP fixed with 4% paraformaldehyde in PBS overnight at 4 °C. The presence of human fibrinogen was probed with anti-human fibrinogen antibodies , whereas human CD41 was probed with anti-human CD41 antibodies . Both antibodies were incubated with their respective targets overnight at 4 °C with agitation before the plate was washed with 0.1% PBST and probed with the respective secondary antibodies. The adhered HUVECs and hVSMCs were first washed with PBS twice and then stained with Mitotracker red  for 2 h at 37 °C. The samples were then washed with PBS before being subjected to fluorescence imaging.Outdated human PRP containing 1 × 10There are no conflicts to declare.RA-008-C8RA01583J-s001


Lactiplantibacillus plantarum-No.122 was applied. It was established that 70.7% of the respondents had never eaten insects and more than 30% would not choose them. However, ECF was suitable substrate for fermentation . In addition, fermentation reduced the total biogenic amines content in ECF (by 13.1%). The highest specific volume showed WB, prepared with fermented ECF . All the tested WB showed similar overall acceptability . However, the highest intensity of emotion “happy” was induced by the WB, prepared with fermented ECF. Most of the WB with non-treated and fermented ECF showed higher acrylamide concentration (except WB with 10% of fermented ECF), in comparison with the control. Finally, fermentation is recommended for ECF inclusion in the main WB formula because fermentation improves not only quality but also reduces acrylamide concentration in WB. The aim of this study was to assess respondents’ opinions on the choice of edible insects as a food, and to evaluate the influence of cricket flour (ECF)  on the quality of wheat bread (WB). Whereas ECF is an additional source of acrylamide precursors, in order to reduce acrylamide formation in WB, fermentation of ECF with  The consumption of edible insects has a long tradition in certain countries . CurrentThe global insect market will steadily grow and the prognosis for the period 2020 to 2027 is that this market is expected to increase by 28.5% . HoweverBread is a widely accepted and consumed food product throughout the world, and its consumption is growing . Wheat bAcheta domesticus) is a good source of protein   were inoculated in 100 g of cricket flour/water .Before the experiment, LAB strain was incubated and multiplied for 24 h in De Man, Rogosa, and Sharpe (MRS) broth culture medium  at 30 °C under anaerobic conditions. A total of 3 mL of fresh LAB grown on MRS broth [average cell concentration of 8.6 logAfterwards, the cricket flour was fermented under anaerobic conditions in a chamber incubator  for 24 h at 30 °C. Non-fermented (/non-treated) cricket flour (mixed with water in the same mass ratio 1:1) was analyzed as a control.Before and after fermentation, pH, color coordinates, LAB viable counts, biogenic amine (BA) concentration, and fatty acid and volatile compound profiles of the samples were analyzed.The pH of the samples was evaluated with a pH meter  by inserting the pH electrode into the cricket flour samples. The color coordinates of the cricket flour were evaluated on the surface using the CIE L*a*b* system .Furthermore, the total color change (ΔE) between the control cricket flour/bread and the rest of the cricket flour/bread samples with 10, 20, and 30% of non-treated or fermented cricket flour was calculated using the equation according to Wrolstad and Smith . The LABSample preparation and determination of the biogenic amines (BA), including tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine, in cricket flour samples was conducted by following the procedure reported by Ben–Gigirey et al.  with somv/v) and fatty acid methyl esters (FAME) were prepared according to Pérez-Palacios et al. [The extraction of lipids for fatty acid (FA) analysis was done with chloroform/methanol  of the cricket flour samples were analyzed by gas chromatography–mass spectrometry (GC-MS). All procedures are described in detail in C—control bread; BCF10; BCF20; BCF30—bread samples with 10, 20, and 30% of cricket flour, respectively; BCF10-F122; BCF20-F122; BCF30-F122—bread samples with 10, 20, and 30% of fermented cricket flour with Lp. plantarum No. 122, respectively). The baking dough was mixed for 2 min at a low speed, then for 6 min at a high-speed regime in a dough mixer . Then, the baking dough was left at (24 ± 2) °C for 15 min relaxation. Afterwards, the dough was shaped into 375 g loaves, formed and proofed at (32 ± 2) °C and 80% relative humidity for 60 min. The bread was baked in a deck oven  at 220 °C for 25 min.The bread formula consisted of 1.0 kg of wheat flour, 1.5% salt, 3% instant yeast, and 1000 mL water (control bread). Control samples were prepared without the addition of non-fermented or fermented cricket flour. The tested bread groups were prepared by addition to the main recipe 10, 20, and 30% of non-fermented or fermented cricket flour. In total, seven groups of dough and bread samples were prepared, and the breads were tested (BAfter 12 h of cooling at (24 ± 2) °C, bread samples were subjected to the analyses of specific volume, crumb porosity, shape coefficient, mass loss after baking, crust and crumb color coordinates, overall acceptability, induced emotions for the trained judges, and acrylamide concentration.FaceReader 8 software , scaling the nine emotion patterns  and valence. The judges were asked to taste the bread samples one by one in front of a Microsoft LifeCam Studio webcam . The recordings were analyzed with FaceReader 8 software and the intensity of facial expressions was expressed on a scale from 0 to 1, as well as valence from −1 to 1. Between the samples, the judges were asked to rinse the mouth with water.Bread volume was established by American Association of Cereal Chemists (AACC) method , and theFinally, the acrylamide concentration was determined according to the method of Zhang et al.  with modn = 3 ± standard error; and for bread overall acceptability and induced emotions with a trained panel, n = 10 ± standard error. In order to evaluate the effects of fermentation and different quantities of cricket flour on bread quality parameters, data were analyzed by one-way ANOVA and Tukey HSD tests as post hoc tests ). I addition, Pearson correlations were calculated between various parameters. The results were recognized as statistically significant at p ≤ 0.05.For the survey interpretation, basic descriptive statistics were used. For cricket flour and bread data interpretation, the results were expressed as the mean values ± standard error: for physicochemical parameters in cricket flour and bread samples, Results of the survey showed that 70.7% of the respondents have never eaten insects as culinary preparation, snacks, or other derived products a. In addMost other studies have revealed that consumers are not very willing to eat insects . Tuccillp = 0.035). According to the values of ΔE, the color of fermented cricket flour was different from the non-treated sample. Correlations between the pH and the color of the samples were not established. Moreover, fermentation was not significant on sample redness (a*). In fermented cricket flour, LAB viable count was 8.24 log10 CFU/g and there was a very strong negative correlation between samples’ pH and LAB viable counts .The pH, color coordinates, ΔE, and LAB viable counts of the cricket flour are given in Lp. plantarum CR L1 strain is suitable for cricket flour fermentation and bread preparation because of the good acidification rates and high LAB viable counts in sourdough [10 CFU/mL) [2); however, the insect flour can lead to a higher buffering capacity of the sourdough [Lp. plantarum strain [The main desirable characteristics for microbial starter cultures (or simply starters) are the acidification rate and the effectiveness of microbial growth in the fermentable substrate . It was ourdough ,44. Howe CFU/mL) . In our  CFU/mL) , while t CFU/mL) . The droourdough . It was m strain . The latm strain .viz.: after 24 h of fermentation on average by 13.0 times and after 48 h of fermentation on average by 18.9 times. A very strong negative correlation between tyramine concentration and samples pH values was observed .Biogenic amine (BA) concentrations in non-treated and fermented cricket flours are given in The highest spermine and spermidine concentrations were found in control samples . The latter BA concentrations in 24 and 48 h fermented samples were on average 144.3 and 167.0 mg/kg, respectively.Yarrowia lipolytica RO25 strain showed the lowest BA content among the analyzed samples [Yarrowia lipolytica RO25 hydrolysate [p ≤ 0.0001). In addition, very strong positive correlations were established between putrescine and spermine, as well as between spermidine and putrescine . Physiologically the most important BA are histamine and tyramine, because of their toxicity [Studies about BA formation in cricket flour during fermentation are scarce. It was reported that the cricket powder hydrolysate by the  samples . For cri samples . It was rolysate . The hearolysate . Despiterolysate . In thistoxicity .cis,trans-9- oleic acid methyl ester , and methyl linoleate (C18:2).The fatty acid profile of non-treated and fermented cricket flour is tabulated in It was reported that the fatty acid profile of crickets consists of oleic acids, linoleic acids, and palmitic acids, with small amounts of arachidonic acid . SimilarVolatile compounds (only when the content of volatile compound was higher than 1% in at least one cricket flour sample are considered) of non-treated and fermented cricket flours are revealed in Lactobacillus plantarum ATCC 8014 strain was mentioned as showing good properties for insect flour fermentation, including sourdough enrichment with fatty acids, amino acids, minerals, and volatile compounds  [The myrcene) . Authorsmyrcene) . The odomyrcene) . In linemyrcene) . Moreove3/g) was found on breads prepared with fermented cricket flour . Similar tendencies were established regarding the bread porosity, that is the addition of non-treated as well as fermented cricket flour increased bread porosity in comparison with control breads (except bread prepared with 10% of non-treated cricket flour). However, the correlation between the specific volume and porosity of bread was not found. In both cases, fermented cricket flour showed better results in comparison with non-treated ones. Significant differences between shape coefficient, moisture content, and mass loss after baking the different groups of bread were not recognized. However, between bread porosity and bread moisture content or mass loss after baking, very strong positive correlations were found . Comparing bread crust and crumb color coordinates, the addition of cricket flour (non-treated and fermented ones) to the main bread formula systematically reduced bread crust and crumb L*, increased crust and reduced crumb a*, and reduced crust and crumb b*. Comparing ΔE, it was found that the bread crust of BCF10-F122 was the most similar to the control bread crust (BC). Indeed, the total color change had proportionally risen from 21.05 to 31.51, and from 14.31 to 23.62, indicating significant differences between bread crust samples with 10% and 30% of non-treated and fermented cricket flour, respectively. It was found that the bread crumb of BCF10 was the most similar to the control bread crumb (BC). Moreover, ΔE had proportionally risen from 8.89 to 14.62, and from 11.56 to 14.42, indicating significant differences between bread crumb samples with 10% and 30% of non-treated or fermented cricket flour, respectively. Bread crust L* and b* coordinates showed positive correlations with bread porosity, moisture content, and mass loss after baking . In addition, bread crumb L* showed positive correlations with bread porosity, moisture content, and mass loss after baking , as well as bread crumb a* and b* showed positive correlations with bread porosity and mass loss after baking . These results showed that not just the colored compounds of the cricket flour but also the changes that are obtained during the thermal treatment have a significant influence on the color parameters of the bread.Bread-specific volume, porosity, shape coefficient, mass loss after baking, ΔE, and color characteristics are given in C, BCF10, and BCF20 samples were not found (on average bread hardness was 0.4 mJ). However, the addition of 30% of non-treated and 10, 20, and 30% of fermented cricket flour increased bread hardness to 0.5, 0.6, 0.6, and 0.7 mJ, respectively. After 48 h of storage samples with 30% fermented cricket flour showed the hardest texture (0.8 mJ). Similar tendencies remained after 72 h of storage; just the bread samples with 10 and 20% of non-treated cricket flour showed the lowest texture hardness (on average 0.6 mJ).The changes in bread texture hardness during storage  are shown in Breadmaking technology is sensitive to substituting wheat flour by gluten-free flour, owing to the decrease of the gluten network . MoreoveOne of the key-quality factor of bakery goods that affects consumer’s acceptability and preference is the color. It was reported that the color difference between control and insect flour-substituted breads was obvious to the human eye . Howeverp = 0.024), as well between the emotion “happy” and bread crust a* a moderate negative correlation was recognized . Other emotions induced in trained judges by bread samples were lower than 0.100, as well as most of the valence of the samples was negative  in bread samples and bread crumb images are tabulated in Lactiplantibacillus plantarum No. 122 strain can be recommended as a good mean to reduce acrylamide formation in bread samples.It was stated that the addition of cricket flour to the main wheat bread formulation led to changes in the crust color, which could be related to a higher intensity of the Maillard reaction. As previously referred, the latter is associated with the formation of aroma and color compounds, as well as with a higher concentration of acrylamide in the end product. Indeed, the aspartic acid and asparagine content in cricket flour can reach 7.52% , and the10 CFU/g and pH 4.26. The total biogenic amine content in fermented cricket flour was 13.1% lower in comparison with non-fermented ones. The main fatty acids in cricket flour were methyl palmitate, cis,trans-9-oleic acid methyl ester, and methyl linoleate, and the main volatile compounds in non-treated cricket flour were acetic acid, hexanal, and decane. Fermented cricket flour  increased bread-specific volume. Significant differences between all bread samples in overall acceptability and the induced emotion “neutral” were not found. However, the highest intensity of the emotion “happy” was induced by the wheat bread prepared with fermented cricket flour. Most of the wheat bread formulations with non-treated and fermented cricket flour showed higher acrylamide concentrations, in comparison with the control bread samples. Finally, it can be stated that the inclusion of cricket flour in the main wheat bread formula leads to better quality characteristics of the baking product but, in most cases, increases acrylamide concentration (except in wheat bread prepared with 10% fermented cricket flour). According to the results obtained, fermentation of cricket flour before its use in wheat bread preparation is recommended as a pre-treatment because fermentation increases not only the quality but also the safety characteristics of wheat bread.In Lithuania insects are non-traditional food and results of the survey showed that more than 30% of the respondents do not want to choose them as food. For this reason, incorporation of the cricket flour into the main traditional food formulations becomes relatively attractive. However, additional proteinaceous ingredients in bread formula could lead to the higher formation of acrylamide. This study showed that the use of pre-treatment for cricket flour with selected LAB strain is an appropriate technology to reduce acrylamide formation in wheat bread enriched with cricket flour. Cricket flour was a suitable substrate for LAB fermentation: in 48 h fermented cricket flour LAB viable counts were 8.24 log


Manihot esculenta Crantz) products: native starch powder (NS), fermented starch powder (FS), and starch granulate (SG). The analyzed properties were compared based on the statistical evaluation of differences in the course of sorption isotherms and the identification and comparison of parameters in two theoretical models of sorption. Empirical data were generated by means of the static-desiccator method. Measurements were made using AquaLab apparatus. The size, shape, and number of tapioca particles were characterized using a Morphology automatic particle analyzer. The study demonstrated that in-depth exploration of empirical data describing hygroscopicity of samples with the use of mathematical tools allows evaluating their physical parameters. The results obtained were analyzed in terms of correlations between physical and physicochemical properties determining utility traits of cassava starch. The NS and SS featured significantly higher hygroscopicity than SG, as evidenced by the values of all parameters analyzed in this study. The study results provided new information related to the management of the production process, safety, and stability of these products.This study aimed to compare hygroscopicity properties of three cassava ( Manihot esculenta Crantz), belonging to the Euphorbiaceae family, is a highly popular crop in the tropical zones of the globe, likewise, e.g., wheat in European countries [Cassava  roots are used to manufacture various starch products with a different fraction size composition, the common feature of which is a high dehydration degree. Water content reduction enables a radical decrease in water activity, and by this means, achieves enzymatic and microbiological stability, which, in turn, ensures adequate storage stability [Cassava  in each of the three mentioned forms. However, starch powder and granules are the most common in the European market, whereas fermented starch cannot be commonly purchased in Poland yet. Tapioca starch (non-fermented) is used as, e.g., a stabilizer or a thickener due to its favorable technological properties and very neutral or utterly neutral taste. Tapioca-based food products are recommended to persons susceptible to allergens, especially those allergic to wheat and/or maize proteins. In addition, the starch of this type is increasingly often applied in food products intended for children, owing to its beneficial physical properties, texture, stability, and neutral taste. However, the most popular food products available on the European market are Asian-style noodles, Asian-style crispy bread, gluten-free bread, or the very popular molecular caviar . Tapioca can also be used to prepare, e.g., pancakes, cakes, or puddings ,16,17.Edible cassava starch, i.e., tapioca, is also used in pharmaceutical products as an excipient and a filling substance. In the native form, it is a constituent of many pharmaceutical formulations—a binder, disintegrant, and a diluent of the active substance. In turn, the modified tapioca starch has an even more comprehensive range of applications, depending on the type of modification and intended use, etc. .Carbohydrates are the most important and, at the same time, the dominant component of each starch, determining its technological usefulness as well as the stability of functional properties and consumer safety. In particular, attention is paid to the ratio of amylose to amylopectin, which in tapioca is at the level of 17–24% amylose and 76–83% amylopectin . The ratManihot esculenta Crantz) starch products , by comparing their sorption isotherms and the parameters of selected mathematical models used to describe these isotherms. The use of the BET  and GAB  models was conditioned by their theoretical nature, allowing for a physical interpretation of the parameters of these models. Moreover, its goal was to identify selected parameters describing the microstructure of the tested products. The study also assessed the differentiation of particle size and shape distributions of the studied starch samples. It was assumed that the analysis of the differentiation of the hygroscopic properties of starch, taking into account the variability of selected physical parameters of these starch particles, will be a source of new valuable information for the management of the production process, safety, and stability of these products.This study aimed to compare the sorption properties, in terms of hygroscopicity, of three cassava (Manihot esculenta Crantz) starch, also called tapioca, were studied: native starch powder (NS), starch granulate (SG), and fermented starch powder (FS). All three starches were produced under industrial conditions. Native tapioca powder (NS) and tapioca granulate (SG) were purchased in Poland, whereas fermented starch (FS) was imported from Brazil. Native tapioca powder was produced in Thailand and distributed in Poland by De Care Group Sp. z o.o. i wsp. Sp. Kom. Tapioca granulate was produced in Thailand by THAI WORLD IMPORT & EXPORT Co., Ltd.  and distributed in Poland by KK Polska Sp. z o.o. Fermented tapioca was produced in Brazil and distributed by SUCOS DO BRASIL—PRODUCTOS LATINO GMB  under the trademark YOKI™. When opened, the experimental material was kept in a tightly closed package, at room temperature, according to producers’ recommendations.Three products made of cassava . The analyzer enables determination of the size distribution of solid particles, with sizes ranging from 0.5 to 10,000 µm. Estimations were made for value distributions of such parameters as: diameter, circularity, convexity, elongation, shape coefficient, and solidity .Water content was determined according to the Polish Standard (PN-ISO 712:2002) in the AquaLab apparatus series 4 model TE , exact to ±0.003, at a temperature of 20 ± 1 °C.wa) range from 0.07 to 0.98, and the time (30 days) required to reach the dynamic equilibrium between the analyzed samples and saturated solutions of respective substances was: NaOH·H2O (0.0698); LiCl·H2O (0.1114); KC2H3O7·1.5H2O (0.231); MgCl2·6H2O (0.3303); K2CO3·2H2O (0.440); Na2Cr2O7·2H2O (0.548); KJ (0.6986); NaCl (0.7542); KCl (0.8513); KNO3 (0.932); and K2Cr2O7 (0.9793). Equilibrium water contents were determined based on the initial masses of the analyzed samples with established water content, and then their changes triggered by the incubation process in desiccators. The values achieved allowed plotting of the isotherms of water vapor sorption in the tested range of water activities. Each point on each of the plotted isotherms was the arithmetic mean from three parallel determinations. Differences in the course of the sorption isotherms in the entire wa range were analyzed statistically using the Student’s t-test of differences between mean values for dependent variables. Differences were considered statistically significant at p < 0.05.Sorption isotherms were plotted using the static-desiccator method at ambient temperature of 20 ± 1 °C, in a water activity (BET Equation (1):v—adsorption, g H2O/100 g d.m.;mv—maximal adsorption value corresponding to the complete coverage of the surface with a monomolecular layer of the adsorbate, g H2O/100 g d.m.;BETc—energy constant BET, describing the difference between the chemical potential of crude adsorbate molecules and those in the first adsorption layer, kJ/mol;wa—water activity at the adsorption temperature [perature .The course of sorption isotherms was analyzed mathematically using the GAB Equation (2):v—adsorption, g H2O/100 g d.m.;mv—maximal adsorption value corresponding to the complete coverage of the surface with a monomolecular layer of the adsorbate, g H2O/100 g d.m.;GABc—energy constant GAB, describing the difference between the chemical potential of adsorbate molecules in the first adsorption layer and higher layers, kJ/mol;k—constant correcting properties of multilayer molecules compared to the liquid phase;wa—water activity at the adsorption temperature [perature .The analysis also used the BET and GAB models consisted of determining the maximal adsorption value corresponding to the complete coverage of the surface with a monomolecular layer of the adsorbate, called the monolayer (mv). Estimation of the monolayer allowed computing of the specific adsorption area (spa) using Equation (3):spa—specific sorption area, m2/g;N—Avogadro number, 6.023 × 1023 molecules/mol;M—molecular weight of water, 18 g/mol;ω—water cross-section area, 1.05 × 10−19 m2/molecule [molecule .The characterization of the sorption properties, in terms of hygroscopicity, using the BETc and GABc were determined, and the k constant in the GAB equation was estimated.In addition, energy constants V—molar volume of the liquid, g/mol;σ—surface tension of the liquid, N/m;R—universal gas constant, J/(mol K);T—temperature, K;cr—capillary radius, nm [dius, nm .Sizes and volumes of capillaries of the examined starch samples were determined for the area of capillary condensation using Kelvin’s Equation (4), assuming the cylindrical shape of the capillaries.BET equation parameters were identified based on empirical data in the water activity range of 0.07 < wa < 0.50 [GAB equation parameters were identified based on empirical data from the entire wa range studied [The w < 0.50 . In turn studied .The statistical significance of differences between the mean water content and water activity in the tested samples was measured using the ANOVA test and the Tukey post hoc test.BET and GAB equations were determined using a numerical algorithm based on non-linear regression and a Monte Carlo algorithm. Minimizing the residual sum of squares (RSS) was adopted as the target function. Standard errors of the equations’ determined parameters were estimated by a numerical algorithm using the SolverAid macro command, which reflects estimates of uncertainty of values of parameters obtained from Solver [RSS) Equation (5):RMS) error Equation (6) expressed in % [N—number of data;ev—experimental equilibrium water content, g H2O/100 g d.m.;ov—predicted equilibrium water content, g H2O/100 g d.m. [0 g d.m. ,29.Parameters of m Solver . Finallysed in % ,28:(6)RMAll computations were made in Excel 2019.The first stage of the study involved a comparative analysis of the characteristics differentiating the parameters of cassava starch particles. This study stage aimed to identify the most effective applications of the starch products tested. In this case, the knowledge of physical properties is the second indicator, after the characterization of biological and chemical properties, enabling the rational management of the production process involving cassava starch, e.g., in food or pharmaceutical industries.Hygroscopic properties represent a cumulative effect of many factors determining the affinity to water molecules . These fIn terms of physical properties, the particles of the analyzed starches differed mainly in size, which was compared based on diameter. The fine-powdered starches (NS and FS) were polydisperse systems differing significantly in their particle diameters but with normal distributions in the values of this parameter. The diameter of fermented starch (FS) particles was greater than that of native starch (NS). This observation was also confirmed by higher values of solidity parameter recorded in the case of FS starch. In turn, the particles of starch granulate (SG) had significantly greater diameters than the two other starches and featured a bimodal distribution of the values of this parameter. The particles of the fine-powdered starches (NS and FS) showed greater variability in their shape regularity. Although the mean values of the circularity parameter pointed to their more remarkable similarity to a square (0.871 ÷ 0.895) compared to starch granulate (SG) particles (0.801), the comparison of the range of circularity values indicated the opposite. Among the particles of the fine-grained powders, there were also perfectly circular particles (1.000) as well as those not circular at all (0.028 ÷ 0.036). In contrast, there were no perfectly circular particles (0.965) of the starch granulate, but even the least circular particles of this starch type were more regular (0.417) than those of the fine-powdered starches (NS and FS). Analogous observations were made for the convexity values of the cassava starch particles. The particles of fine-powdered starches (NS and FS) were similar in terms of distribution of convexity value. In contrast, the starch granulate (SG) particles were characterized by greater clustering of their values manifested in a narrower range of the extreme values. By analogy, the values of the parameter described as elongation fitted within a substantially broader range in the case of SG particles compared to the NS and SS particles. As a consequence, the shape coefficient values of SG particles also fell in a significantly narrower range than those determined for the particles of the two other starch types. It may be concluded that the starch particles in the form of granules (SG) differed significantly from those of the fine-powdered starches (NS and FS), which also differed significantly in their sizes (diameter and solidity). The starch granulate (SG) differed significantly in terms of its particle characteristics from the two other starches (NS and FS). It may also be concluded that the fermentation process applied to produce fermented starch modified the size of its particles, including their diameter and solidity values. In contrast, it did not modify the shape, circularity, convexity, or elongation of cassava starch particles .A comparison of native cassava starch (NS) to other starches shows that, in terms of the diameter of its particles, it is very similar to native potato starch. However, its particles are more solid (massive) than those of potato starch .The hygroscopic properties represent a cumulative effect of: interactions between body surface and water, water vapor condensation in capillaries, concentration and type of water-soluble substances, and water content of a product . Water sData collated in crit. = 0.868; TNS/SG = 4.2417; TNS/FS = 0.8223; TSG/FS = 3.4193). On the other hand, the assessment of the differentiation in the water activity level showed statistically significant differences between all the samples .The statistical assessment of the significance of differences between the mean values of water content and activity performed with the ANOVA test and the Tukey post hoc test (T) showed that only native and fermented starch did not differ significantly in terms of water content  differed significantly from the other starches in terms of its position in the reference frame. Such a position of the isotherm indicates that, during storage of the analyzed starches in the environment with a specified humidity level, starch granulate (SG) will adsorb significantly less water reaching the same level of its activity as the other two fine-powdered starches (NS and FS). In addition, if all analyzed starches have the same water content, the starch granulate (SG) will feature the highest water activity. This, in turn, will make it the least stable, primarily regarding microbiological safety. The most likely reason behind the significantly different hygroscopic properties of SG is its smaller specific surface area compared to the developed surface of fine-powdered starches.wa ≈ 0.7. Suriyatem and Rachtanapun [The second inflexion on the sorption isotherms of all starch samples was observed at htanapun  reportedhtanapun . This phhtanapun .wa range was also compared in the statistical analysis using the Student’s t-test for differences between mean values for dependent pairs [SN/FS = 3.3096, tSN/SG = 6.7958, tFS/SG = 6.9045, tcrit. = 2.228). The determined statistical values confirm a huge difference between the hygroscopic properties of starch granulate (SG) and those of the other two starch samples. In addition, they show that the fine-powdered starches (NS and FS) also differed in their hygroscopicity. The above results allow concluding that both starch fermentation and compression into compact granules (granulation) lead to significant differences in its hygroscopic properties. This is an important finding, as certain works demonstrate a paucity of data that would confirm the correlation between the physical properties (size and shape) and hygroscopic properties of powders or small-sized solid bodies [The course of sorption isotherms in the entire nt pairs ,31. The d bodies . The resd bodies  showed td bodies .BET and GAB [BET model is most frequently employed to describe the structure of a product and sorption phenomena. It assumes that the shape of a sorption isotherm is due to the complex character of the sorption process on porous bodies [wa values [BET equation of the analyzed cassava starches.The empirically determined sorption isotherms were described using two theoretical mathematical models:  and GAB . The BETs bodies . Using ts bodies . When maw values . Table 3BET model was evaluated based on the residual sum of squares (RSS) and root mean square (RMS) errors. The comparison of the RSS values shows that the BET model described empirical data of all starches with similar accuracy. In turn, the RMS value determined for SG exceeded the threshold of 10%, indicating a good fit of the model to experimental data [BET model better describes the hygroscopicity of fine-grained powders than granules; hence, the results characterizing the fine-grained powders can be claimed to be more reliable.The goodness of fit of empirical data to the results generated using the tal data . This memv) describes the sorption capacity of the adsorbent and the availability of polar sites to the water vapor [2O per 100 kg dry matter. The mv values estimated for cassava starches fitted within this range. The mv values estimated for individual starches differed, indicating that both cassava starch fermentation and granulation affected its water-binding capability. The fermentation process modified the physical parameters of native, which means a crystalline form of starch, having sparse amorphous regions due to peripheral damage of starch granules [The monolayer  and extrapolates data obtained at different temperatures [The second model tested in the study was the ologists . The GABsorption ,48. It eeratures . Due to GAB equation at ±15.5% required the k constant to fit the range of 0.24 ÷ 1, and the GABc constant to be higher than 5.67 [RSS and RMS values indicate that the GAB model proved very good in describing water vapor sorption by the analyzed starch samples. Model parameters were calculated for each starch type with the same accuracy. In turn, a comparison of RMS values shows that the GAB model proved better than the BET model in describing the sorptive properties of cassava starches [Lewicki demonstrated that maintaining the calculation error of the monolayer estimated based on the han 5.67 . These cstarches .GAB equation were higher than those determined with the BET equation; however, the distribution of GAB model results was analogous to that achieved with the BET model. Native starch (NS) had the largest monolayer (11.2), whereas starch granulate (SG) had the smallest monolayer (8.7), which was most likely due to the modifications in the physical structure of starch granules [The monolayer values estimated using the granules  caused bGABc was defined as a difference between the enthalpy of adsorbate molecules in the first adsorption layer and the higher layers [GABc value [The energy constant r layers . In turnAB value . Given tk parameter also describes the deviation between desorption enthalpy and liquid adsorbent evaporation enthalpy and corrects the properties of molecules building the monolayer compared to the liquid phase. Such a deviation does not occur only when k equals 1 [k value enables distinction between monolayer (k ≤ 0.5) and multilayer (k > 0.5) adsorption. For this reason, the determined values of k make the GAB model a reliable tool for describing the sorption properties of the analyzed starches. Furthermore, the k constant values determined for individual starch types were similar, which explicitly confirms the similarity of the examined material (cassava starch), which has earlier been pinpointed by Chirife and Iglesias [The equals 1 . In addiequals 1  claims, Iglesias . TherefoIglesias .The last stage of the study aimed to determine selected parameters describing the microstructure of the surface of the analyzed starches. The results obtained indicate that the native (NS) starch, and therefore semicrystalline starch, was characterized by the most developed specific sorption surface area. As a result of the fermentation process and granulation, the specific surface of the starch decreased. This change, however, cannot be associated with an increase in the ordering of starch particles, but only with their physical modifications. Starch fermentation resulted in the increased solidity of its particles. However, as a result of granulation, all parameters describing the physical state of starch particles changed radically  . MoreoveCassava starch-based products can complement the assortment of starch products available on the Polish market. Native cassava starch is similar to native potato starch in terms of particle characteristics but significantly different in this aspect from native wheat gluten-free starch and native maize starch .Edible cassava starches, regardless of their type , showed strong hygroscopic properties , which wBET and GAB models to identify the parameters of hygroscopicity characteristics.Sorption isotherms plotted for all tested cassava starches  were sigmoidal and continuous over the entire range of water activities. This finding justified using BET and GAB) used to explore experimental data were based on well-established theoretical foundations [RSS and RMS values, and by errors with which the parameters of both equations were estimated. However, taking into account the recommendations of the European COST 90 Project [GAB model, the possibility of transferring the results obtained using the GAB model to different temperatures, and slightly better results of this model fitting to the original data, it can be concluded that the GAB model should be recommended for the description and comparison of the hygroscopic properties of all tested cassava starches.The mathematical sorption models (ndations . These m Project , a broadBET model, despite similar particle size characteristics.Native cassava starch significantly differs from potato starch in terms of hygroscopicity described by the parameters of the Modification of cassava starch, both through fermentation and granulation, significantly modifies its physical and hygroscopic parameters.Modification of physical/chemical properties, and consequently hygroscopic properties of cassava starches, opens avenues for its more comprehensive and targeted use in both the food and pharmaceutical industries.Modification of cassava starch may contribute to the rational management of the food or pharmaceutical production processes, considering the diverse needs of these industries and consumers.The analysis of the results obtained using both models indicates that:


An average of 3,280 recovered deceased donor kidneys are discarded annually in the United States. Increased cold ischemia time is associated with an increased rate of organ decline and subsequent discard. Here we examined the effect of prolonged cold ischemia time on kidney transplant outcomes.Retrospective observational studyRecipients of deceased donor kidney transplants in the United States from 2000 to 2018.Recipients of deceased donor kidneys were divided based on documented cold ischemia time: ≤16, 16-24, 24-32, 32-40, and >40 hours.The incidence of delayed graft function, primary nonfunction, and 10-year death-censored graft survival.The Kaplan-Meier method was used to generate survival curves, and the log rank test was used to compare graft survival.The rate of observed delayed graft function increased with cold ischemia time . Primary nonfunction also showed a similar increase with cold ischemia time , During a median follow-up time of 4.6 years, 37,301 recipients experienced death-censored graft failure. Analysis based on kidney donor profile index (KDPI) demonstrated significant differences in 10-year death-censored graft survival, with a death-censored graft survival in recipients of a kidney with a KDPI <85% of 71.0% , 70.5% , 69.6% , 65.5% , and 67.2% , compared to 53.5% , 50.7% , 50.3% , 50.7% , and 48.3% , for recipients of a kidney with a KDPI >85%.Heterogeneity of acceptance patterns among transplant centers, presence of confounding variables leading to acceptance of kidneys with prolonged cold ischemia times.Cold ischemia time was associated with an increased risk of delayed graft function and primary nonfunction. However, the effect of increased cold ischemia time is modest and has less impact than the KDPI. Transplant programs should not consider prolonged cold ischemia time alone as a predominant reason to decline an organ, especially with a KDPI <85%.  One of the main considerations in whether to accept or decline a kidney is the amount of time that has passed since the kidney has been recovered, also known as cold ischemia time. As the time the kidney has been out of the donor increases, there are concerns that it develops irreversible ischemic injury and is no longer viable. We sought to determine if very long cold times resulted in inferior kidney transplant graft survival. We examined the outcomes of kidney transplants with different cold ischemia times and found that as cold time increased, kidney transplant outcomes were worse. However, this effect was small, and more important to graft outcomes was the quality of the kidney.Kidney transplantation is considered the optimal therapy for eligible candidates with kidney failure. Despite the clear advantages of transplantation in terms of life expectancy, quality of life, and overall health care savings, not all candidates experience its benefits. For the past 5 years, the overall number of kidney transplants has increased annually, yet there are still over 90,000 candidates awaiting transplantation. The average waiting time for a kidney transplant is 3.4 years for first-time listed candidates and 4.3 years for repeat transplant candidates and varies according to blood type and the geographic location, such that waiting time may reach more than a decade for some candidates.In December 2019, to achieve broader and more equitable sharing of deceased donor kidneys and minimize the impact of geography on organ allocation, the board of the Organ Procurement and Transplantation Network approved a proposal to prioritize deceased donor kidney offers for candidates listed at transplant hospitals within a 250-nautical mile radius of the donor hospital. This proposal replaced the use of local donor service areas and regions in the allocation algorithm.The Organ Procurement Transplantation Network/United Network for Organ Sharing database as of September 2020 was used in this study. All deceased donor kidney transplants (DDKTs) from January 2000 to December 2018 were included. Kidneys are classified according to the kidney donor profile index (KDPI), which combines 10 donor factors to form a metric of quality for deceased donor kidneys relative to others recovered. Kidneys with KDPI ≥85% are projected to have reduced graft survival when compared to kidneys with 0%-85%.Multiple organ or double kidney DDKTs, DDKTs with missing data for the KDPI or CIT were excluded. This study was institutional review board exempt because of its use of publicly available data and absence of identification of individual donors and recipients. We grouped DDKTs based on documented CIT as follows: ≤16, 16-24, 24-32, 32-40, and >40 hours.The study population was analyzed to determine the graft outcomes of longer CIT kidney utilization. The primary outcome of interest was death-censored graft survival, defined as the time from transplant to the earliest of allograft loss, kidney retransplantation, reinitiation of dialysis, or loss to follow-up with a functioning graft, censored for death. Secondary outcomes included delayed graft function (DGF), defined as dialysis within the first week post-transplantation, primary nonfunction (PNF), defined as permanent loss of allograft function starting immediately after transplantation, and serum creatinine at the first year posttransplant.2 test as appropriate. Serum creatinine levels at the first year posttransplant were only available for individuals who were alive, being followed clinically, and did not experience graft failure at 1-year follow-up. The Kaplan-Meier method was used to generate survival curves, and the log rank test was used to compare graft survival between groups. Because donor kidney quality also affects graft outcomes, we separated survival curves into KDPI <85% and ≥85%. In regression analysis, CIT was modeled with restricted cubic splines with 4 knots located at 16, 24, 32, and 40 hours. Cox proportional hazards model was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) to examine risks associated with graft loss. A logistic regression model was used to calculate odds ratio (OR) and 95% CI to examine risks associated with DGF and PNF. CIT of 16 hours was used as a reference. Because of a low number of DDKTs with a CIT >48 hours (0.5%), we grouped donors with a CIT ≥48 hours together in the regression analysis plots. In the multivariable model, we adjusted for (1) donor sex, smoking, cytomegalovirus status, and KDPI; (2) recipient age, sex, African American ethnicity, diabetes, cytomegalovirus status, hepatitis C virus status, and previous kidney transplant; and (3) transplant variables, which included machine perfusion pump used, panel reactive antibody, human leukocyte antigen mismatch, and induction immunosuppression. STATA version 13 (Statacorp) was used in all statistical analyses.Donor, recipient, and transplant characteristics were evaluated. Variables were analyzed using median and interquartile range for continuous variables and proportions for categorical variables. Demographic differences and posttransplant outcomes related to kidney allograft between groups were compared using Kruskal-Wallis or Pearson’s χThere were 223,429 DDKTs between January 1, 2000 and December 31, 2018. We excluded 44,082 DDKTs from the study . A total of 179,347 DDKTs were included in our analysis. Median follow-up time was 4.6 (0-17.9) years. Among these, 50,704 (28.3%) recipients died and 37,301 (20.8%) recipients experienced death-censored graft failure. DDKTs were divided into 5 groups based on documented CIT: ≤16, 16-24, 24-32, 32-40, and >40 hours, representing 83,870 (46.8%), 59,782 (33.3%), 25,017 (14.0%), 7,377 (4.1%), and 3,301 (1.8%) recipients, respectively. Baseline donor, recipient, and transplant characteristics are shown in The overall incidence of DGF was stable during the study period but was higher among longer CIT groups: 17,538 (20.9%), 16,776 (28.1%), 8,101 (32.4%), 2,767 (37.5%), and 1,182 (35.8%) among CIT ≤16, 16-24, 24-32, 32-40, and >40 hours, respectively . CompareThere was a steady decrease in the overall incidence of PNF between 2000 and 2015; however, the rate was stable in the years 2015-2018 at 0.6%, 0.9%, 1.3%, 2.1%, and 2.3% among CIT ≤16, 16-24, 24-32, 32-40, and >40 hours, respectively , Fig S2.There were no clinical differences in median serum creatinine at the first year after DDKT, which were 1.3 mg/dL , 1.3 mg/dL , 1.4 mg/dL , 1.4 mg/dL, and 1.4  mg/dL among CIT ≤16, 16-24, 24-32, 32-40, and >40 hours, respectively .Among donors with a CIT <16, 16-24, 24-32, 32-40, and >40 hours, respectively, 16,592 (19.8%), 12,571 (21.0%), 5,533 (22.1%), 1,803 (24.4%), and 802 (24.3%) recipients experienced death-censored graft failure during the follow-up time. Ten-year overall death-censored graft survival rates were 70.1% , 69.1% , 68.1% , 63.9% , and 65.5% , respectively. There were 12,493 DDKT recipients (7%) receiving a kidney with KDPI ≥85%. When kidney quality was categorized using the KDPI, 10-year death-censored graft survival rates among CIT groups with a KDPI <85% were 71.0% (70.5-71.5), 70.5% (69.9-71.0), 69.6% (68.7-70.4), 65.5% (63.7-67.3), and 67.2% (64.6-69.6), respectively. If KDPI was ≥85%, 10-year death-censored graft survival rates were 53.5% (51.1-55.8), 50.7% (48.3-53.1), 50.3% (46.6-53.8), 50.7% (45.1-56.1), and 48.3% (40.0-56.1), respectively . CompariDuring the study period, United Network of Organ Sharing did not provide the specific causes of organ discard. However, these data were available from the OneLegacy Organ Procurement Organization that recovers approximately 10% of all deceased donor kidneys in the United States. Out of a total of 3,500 deceased donor kidneys that were recovered by OneLegacy between 2015 and 2019, 295 (8.7%) were ultimately discarded because of prolonged CIT alone or prolonged CIT along with other factors.Increased CIT is associated with an increased rate of organ decline by transplant centers and consequent organ discard. Our study confirms the concerns surrounding increased CIT and organ transplant outcomes as DGF and PNF increased with each CIT interval studied. In a multivariate regression analysis adjusted for recipient and donor characteristics including the KDPI, a longer CIT >24 hours was associated with an increased risk for DGF, PNF, and eventual graft failure, an observation that has been previously reported.Serum creatinine values 1 year post kidney transplantation has been shown to be one of the best predictors for long-term kidney allograft survival. In this study, overall median serum creatinine at 1 year posttransplant did not change with increasing CIT interval (1.3 mg/dL), and a survival analysis showed comparable 10-year death-censored graft survival between CIT <16 hours and the longer CIT groups. Although CIT duration did not result in observable differences in 1 year serum creatinine, there were some key differences in each group, including higher rates of donor hypertension, donor diabetes, and terminal creatinine. One striking observable difference between the groups was the increased use of machine perfusion with increasing CIT group, with 26.9% in those with CIT <16 hours and 79.6% in those with CIT >40 hours. The effect of machine perfusion on reducing DGF/PNF and in resistance indices predicting DGF and discard have been well documented. It is possible that the use of machine perfusion provided additional information in the acceptance of organs with increased CIT or provided a milieu for limiting additional injury to the transplanted organ. Additional studies are necessary to determine the potential confounding and/or beneficial effects of machine perfusion on organs experiencing prolonged CIT.Organ quality, as determined by KDPI, has an effect on long-term transplant survival. Higher KDPI organs, which represent lesser quality organs, are more likely to have a higher serum creatinine 1-year posttransplant and lower long-term graft survival, which our study also confirmed. CIT has a negative potentiating effect in combination with KDPI, resulting in additionally worse outcomes with higher rates of DGF and PNF. However, in those transplants that functioned, CIT interval did not correlate with adverse outcomes. As expected, our study demonstrates that CIT in high-quality organs outperformed those with a KDPI >85%. Unexpectedly, we found that those organs with a CIT >40 hours with KDPI <85% performed better than those with minimal CIT and a KDPI >85%. This is likely driven by of our finding that 1-year serum creatinine was unrelated to CIT and the much lower incidence of PNF seen in the KDPI <85% group. However, the low number of organs transplanted in this group may limit this conclusion. This suggests that lower KDPI organs can better endure ischemic injury and recover from DGF without long-term detrimental effects to long-term graft function.One clear need arising from our results is the ability to better predict the risk for PNF, as our data clearly demonstrates that if the organ is functional, short and long-term graft survival is comparable across CIT exposure. However, the potential impact of utilizing high-quality organs with prolonged CIT should not be ignored, especially in light of recent organ allocation changes. An inevitable consequence of the goal of reduction in the geographic disparities in organ allocation is an increase in CIT, as deceased donor kidneys are more likely to be transported over longer distances and across organ procurement organizations. During the study period national data for specific reasons for discard were not provided. However, utilizing data from OneLegacy Organ Procurement Organization, which recovers approximately 10% of all deceased donor kidneys in the United States, revealed that 8.7% of kidneys were discarded for prolonged CIT alone or with other factors. Extrapolating the OneLegacy organ acceptance data highlighting the role of CIT as a contributing factor for organ decline nationally, utilizing organs with longer CITs could increase the number of overall transplants by several hundred each year.The primary limitation of our study is the lack of information on discarded organs. It is unclear if the outcomes reported are more favorable because of careful organ selection and/or the presence of additional mitigating factors not documented in the database. The low number of patients at risk in the CIT group >40 hours in recipients with a KDPI >85% also limits conclusions for this group.In conclusion, kidneys with prolonged CIT, which are frequently discarded, may provide years of dialysis independence for patients on the transplant waiting list. Transplant programs should not consider prolonged CIT as a predominant reason to decline an organ, a recommendation that is particularly relevant when kidneys are transported beyond the 250-nautical mile radius.


Reviewers 2021Recenzenci 2021Editorial Board of the Journal of Mother and Child / Medycyna Wieku Rozwojowego would like to thank all the Reviewers for their time and commitment to the evaluation of the works.Redakcja Journal of Mother and Child / Medycyna Wieku Rozwojowego składa serdeczne podziękowania wszystkim Recenzentom za poświęcony czas i zaangażowanie w oceny prac.