Humans can increase the endpoint stiffness of their arm to reduce self-generated movement variability and to reject unpredictable perturbations from the environment, like during handheld drilling, thereby increasing movement precision. Existing methods to estimate changes in the endpoint stiffness use robotic interfaces to apply position or force perturbations to measure the arm’s dynamic response. We propose an alternative method of measuring changes in the power grasp force to estimate adaptations in the magnitude of the arm’s endpoint stiffness. To validate our method, we examined how the strength of the power grasp, when holding onto a robotic manipulandum, affected the arm’s endpoint stiffness in three different locations of the workspace. The endpoint stiffness magnitude increased linearly with the grasp force, and this linear relationship did not depend on the arm’s posture or position in the workspace. The endpoint stiffness may have increased as a combination of greater grasp stiffness and greater arm stiffness, since larger co-contraction was observed in the elbow and shoulder with a stronger grasp. Changes in the grasp force could serve as a metric in assessing how humans adapt their endpoint stiffness magnitude. This task is unstable as the screwdriver can slip out of the hole during rotation. Humans cannot react fast enough to correct a slipping screwdriver as visual feedback is delayed by approximately 200 milliseconds. Instead, the central nervous system (CNS) adopts a strategy of co-contracting muscle pairs in a joint to increase the endpoint stiffness of the arm. The endpoint stiffness as defined in this study is the stiffness of the upper arm, including the hand, which generates a restoring force that bring the hand to its equilibrium position. The endpoint stiffness is a mechanical property of the arm, such that the restoring force is generated without sensory feedback delays3 and reject perturbations from the environment5. Existing methods to estimate the arm’s endpoint stiffness employ a robotic interface to perturb the arm’s position and measure the restoring force generated by the arm9. Force perturbations can also be employed to estimate the arm’s endpoint stiffness10. These methods require a robotic interface, which are expensive and are not portable, making them unsuitable for measurements outside of the laboratory environment. In a recent study, we reported that when subjects had to reach with higher movement precision, they increased their hand’s power grasp force11, which we define as the force measured between the palm of the hand and the handle of the robotic interface. The adaptation of the grasp force was similar to the manner in which the arm’s endpoint stiffness has been reported to change in an unstable environment12. If the grasp force and the arm’s endpoint stiffness are related, measuring changes in the power grasp force may yield an affordable and portable method of estimating changes in the magnitude of the arm’s endpoint stiffness.Studies have shown that humans are able to adapt the stiffness of their arm to suppress self-generated motor variabilityIn this study, we deliberately chose to measure the endpoint stiffness by exerting position perturbations through the subject’s hand. Some studies use a mold of the hand that is attached to the handle of the robotic interface to perturb the subject’s arm at the wrist, effectively bypassing the hand and the need to grasp the robotic interface. However, the endpoint stiffness of the arm in real-world tasks does not bypass the hand. When using a hammer or a chisel to carve wood, the endpoint stiffness at the tip of the tool depends on both the stiffness of the grasp and the stiffness of the arm. We define the grasp stiffness to be the stiffness in the connection between the handle of a held object and the hand. The stiffness of the arm arises from the muscular co-contraction of muscles in the elbow and the shoulder. The stiffness of the arm alone may not reflect how humans increase endpoint stiffness in real-world tasks. As such, it is important to ascertain the endpoint stiffness at the hand, i.e. the combined stiffness of the grasp and the arm.14. We hypothesized that the magnitude of the endpoint stiffness of the arm was positively correlated with the grasp force, and that the posture may interact with this relationship.The aim of this study was to assess the relationship between the hand’s power grasp force and the magnitude of the endpoint stiffness of the arm. We hypothesized that this relationship may depend on the arm’s posture, and so we measured the arm’s endpoint stiffness as a function of grasp force in three different postures. When measuring the endpoint stiffness of the arm, we opted for a position perturbation paradigm commonly used in the literature where a hand-held robotic interface perturbs the arm’s position by several millimeters and measures the restoring force to estimate the arm’s endpoint stiffness20 subjects, who all gave their informed consent, participated in the study (10 subjects in the first experiment and 10 subjects in the second experiment). The experimental protocol was approved by the Ethical Review Committee for Epidemiological Studies at the Tokyo Institute of Technology (reference number A17086), and was conducted in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The subjects were seated facing the KINARM planar robotic manipulandum from BKIN Technologies see Fig. . A subjeOnce seated, subjects were instructed to keep their head and torso in the same posture throughout the experiment. This was accomplished by having the subjects rest their forehead on a fixed headrest, with the chest pressing against a table placed below the robotic interface. The position of the shoulder, and the lengths of the forearm and the upper-arm were measured prior to the experiment to determine the posture of the arm.t, was controlled to maintain its position at a commanded setpoint Ld = 130 Ns/m.The robotic manipulandum’s position 0 =  m, which are hereby referred to as the left, center and right positions, respectively. y0 = 0.12 m was fixed for all three positions. The joint angles for each posture were We measured the arm’s endpoint stiffness in three different positions of the workspace that corresponded to different setpoints xx0. This was accomplished by a small rapid perturbation, corresponding to a fifth order polynomial of the formT = 100 milliseconds. The position perturbation started from zero when t = 0 and reached an amplitude A when t = T.To estimate the arm’s endpoint stiffness, it is necessary to physically perturb the arm’s position from the resting position xp(t) was rotated by a rotation matrix x0 to perturb the arm’s position in eight different directions. After the 100-millisecond position perturbation ramp, the perturbation The position perturbation x-axis. In this plot, dx = 0. The shaded areas denote where the restoring force was measured. The velocity of the endpoint in the shaded area was effectively null. All force data was low-pass filtered using a zero-lag, second-order Butterworth filter with a cutoff frequency of 20 Hz. The change in the filtered force signal along the x-axis dFx, which is plotted in the bottom panel of Fig. Figure 15. The visual feedback was restored between each perturbation. Once the eight perturbations were finished, subjects were instructed to relax for 5 seconds, after which another target grasp force was presented.Prior to the start of the experiment, the experimenter instructed the subject to maintain a specific level of grasp force as shown on the monitor. A red circle displayed the target grasp force that subjects had to produce and maintain. The applied grasp force was displayed by a filled white circle that grew in size. Once the subject held the target grasp force within a tolerance of ±1 N for 300 milliseconds and was applying less than 1.5 N of load force against the robot, the robot began to perturb the subject’s arm in eight directions chosen at a random order. Subjects were instructed to ignore the position perturbations and focus only on maintaining the target grasp force. During the position perturbation period, which lasted 600 milliseconds, the feedback of the hand’s position and the applied grasp force was frozen to the last value measured prior to the position perturbation. This ensured that subjects did not exhibit correctional movements driven by visual feedback during the perturbationThe target grasp force, which ranged from  N, was selected in a random order. The order in which the endpoint stiffness was measured for the three different postures was fixed for all subjects. First, subjects matched all four grasp force levels in the left posture, and then the center posture, and finally the right posture. This process was repeated again for a total 3 cycles such that each posture and each grasp force level was tested three times to have a robust estimate of the endpoint stiffness, and to assess the consistency of our measurements. Hence, 24 perturbations were measured at each posture and each grasp force level per subject.M, viscosity D and stiffness K of the arm were linear7 such that the dynamics could be described byF0 is the force measured prior to the perturbation. Once the position of the arm reaches its perturbed steady-state value with null velocity and null acceleration, this equation simplifies toFor small perturbations, we assumed that the inertia dx and dF were measured by the KINARM’s position sensing and its six-axis force sensor. This data was linearly regressed to find the endpoint stiffness K. The data from all perturbations in all trials was used in the analysis to estimate the endpoint stiffness for a given posture and grasp force level per subject.The quantities of 13.For a specific grasp force level and posture, we gathered the data from the 100-millisecond perturbation window  was measured using the Delsys Trigno wireless EMG sensor system at 1000 Hz from the elbow monoarticular pair ; the biarticular pair (biceps brachii and the triceps long head); and the shoulder muscle pairs were measured . In this experiment, subjects also wore a wrist brace to prevent wrist movements.The mean EMG activity in the same 100-millisecond window, where the restoring forces were measured, was calculated for each perturbation.s, b and e denote the shoulder, the biarticular and the elbow, and the positive and negative subscripts indicate the flexor and extensor muscle, respectively. Under the Kelvin-Voigt muscle model, the stiffness of a muscle is proportional to its muscle activation18, e.g. ub is the sum of the biarticular activity and ue is the sum of the elbow monoarticular muscle pair’s activity. Hence, an increase in the activity of the elbow flexor and/or extensor with greater grasp force would imply an increase in the stiffness of the elbow joint.The arm’s joint stiffness matrix is composed of the stiffness of each muscle in the upper arm, givingj is the repeated measurement index  and k is the grasp force level . Altogether, the averaged activity To compare the activation of the muscles between subjects, the activity of each muscle was normalized by dividing it with its average activity in all trials,We used a two-way repeated-measures analysis of variance to examine the effect of the grasp force and the posture on the size of the endpoint stiffness ellipse. One-sample t-tests with a Holm-Bonferoni correction were conducted on the normalized EMG activity of the elbow, shoulder and cross-joint to find the significance of the linear gradient between the EMG data and the grasp force.x-axis from a sample trial containing two position perturbations. The shaded regions in Fig. dx = 0. For convenience, all subsequent values with errors bars will denote the group mean and the standard error.Figure We examined if the subjects were successful in maintaining the target grasp force during the experiment. The grasp force was averaged in a 100-millisecond window of the perturbation near the end of the position perturbation. This average grasp force was calculated per perturbation direction, and the mean of all directions and postures was taken to find the group mean grasp force for each target level of grasp force. The group mean grasp force, along with the standard error, were found to be 4.79 ± 0.06 N, 9.55 ± 0.09 N, 14.30 ± 0.10 N and 19.14 ± 0.10 N. The grasp force was comfortably within a tolerance of 4% of the target level, implying that the subjects were successful in maintaining the grasp force during the perturbations.Next, we looked at the restoring force in the left, center and right positions from a sample subject  = 0.81, p > 0.57). On the other hand, the repeated-measures ANOVA revealed a significant effect of the grasp force on the size of the endpoint stiffness ellipse  = 43.5, p < 10−9), supporting our hypothesis that the grasp force is positively correlated with the magnitude of the endpoint stiffness. The normalized size of the stiffness ellipse grew by 37% when the grasp force increased from 5 N to 20 N . We also calculated the size of the endpoint stiffness ellipse for each subject as a function of the grasp force, averaging the data over the three different positions. Figure 4 N2/m2 and 105 N2/m2 for our ten subjects, resembling the range observed in other studies7.Table What caused the endpoint stiffness of the arm to increase with the grasp force? We recruited a new set of 10 subjects and measured their endpoint stiffness as a function of the grasp force. In this experiment, six wireless surface electromyography (EMG) sensors were placed on the subject’s elbow monoarticular, biarticular and shoulder muscle pairs (see Methods for selected muscles). The mean muscle activity was measured in the 100-millisecond window where the restoring force from the arm was measured. For this experiment, we only measured the endpoint stiffness in the center position, and each grasp force level was measured five times. Subjects also wore a wrist brace to prevent wrist movements. The activity of each muscle was normalized by dividing its mean activity measured in all grasp force levels (see Methods for details).Table us and the biarticular pair ub contribute to the shoulder joint stiffness Kss, and the elbow joint stiffness Kee is composed of the activity of the elbow monoarticular pair ue and the biarticular pair ub, whilst the activity of the biarticular pair ub contributes to the cross-joint stiffness Kse and Kes. The sum of the normalized activities of these muscles is plotted as a function of grasp force in Fig. t(9) = 2.87, p < 0.019), the elbow joint stiffness (t(9) = 5.86, p < 0.002) and the cross-joint stiffness (t(9) = 3.41, p < 0.008) significantly increased with the grasp force. Thus, the co-contraction in the elbow, shoulder and the cross-joint contributed to the increase in the magnitude of the arm’s endpoint stiffness.The normalized muscle activity from the elbow, biarticular and shoulder muscle pairs are plotted as a function of the grasp force from a sample subject in Fig. We perturbed the position of the arm to estimate the arm’s endpoint stiffness in three different positions of the workspace with different levels of power grasp force. The magnitude of the endpoint stiffness increased with the grasp force. This relationship was observed in the three different postures that we tested, implying that the linear correlation between the grasp force and the endpoint stiffness magnitude was robust under different postures.The restoring force was symmetrically distributed around the origin in the left, center and right positions, implying that for small position perturbations and a minimum grasp force of 5 N, there is little risk of the robotic handle slipping out of the subject’s hand, which may adversely influence the measurement of the endpoint stiffness. Furthermore, the high proportion of the variance explained by the linear regression of the restoring force against the position perturbation shows that a consistent estimate of endpoint stiffness was obtained by our perturbation methodology where visual feedback of the grasp force was provided.20. By measuring the muscle activity of the elbow monoarticular pair, the biarticular pair and the shoulder pair in the second experiment, we found that muscular co-contraction of the elbow, shoulder and cross-joints increased with the grasp force. This synergistic activation has been previously observed between the power grasp and either the elbow or the shoulder muscles in other studies22. The increase in muscle activity was somewhat more pronounced in the elbow monoarticular pair in comparison to the biarticular and the shoulder muscle pair. This suggests that muscles closer to the hand activate more strongly during grasping in comparison to muscles further away from the hand.Our study cannot determine whether the primary contributor to the increase in the endpoint stiffness was the grasp stiffness or the stiffness of the arm. However, the EMG data shows that the increase in the endpoint stiffness was not solely due to greater grasp stiffness, which is known to increase with the grasp force11. The reaching tasks tested in this study were inspired by earlier studies that had reported an increase in endpoint stiffness during their tasks5. In one of these tasks, subjects reached in an unstable force field that pushed the hand left or right when it deviated from the midline. This task required higher endpoint stiffness to precisely move along the midline to reduce the effects of the unstable force field4. The grasp force measured during the learning of this task11 resembled the change in the co-contraction of the arm reported in another study23. Namely, the grasp force increased rapidly over the course of five trials, and then exponentially declined in magnitude and plateaued at a level higher than in training trials without the force field.In another study, we found that the grasp force increased when subjects desired higher movement precision in two reaching tasks4. The loss of the stiffness ellipse’s orientation is traded off by the superior temporal information provided by the grasp force. As was observed in a previous study of ours11, the grasp force changed not only between trials, but within each reaching movement. The change in the grasp force resembled the temporal profile of the movement imprecision when reaching in an unstable force field. EMG also provides a high temporal resolution estimate of the endpoint stiffness of the arm, but it requires calibration at different postures to be effective in estimating the endpoint stiffness during reaching19. The grasp force, on the other hand, was constant during normal reaching and was not influenced by the load force nor by external forces imposed by a robotic interface11. Thus, analyzing the changes in the grasp force may further our understanding of the adaptation in endpoint stiffness during both postural and reaching tasks.Although the grasp force methodology may signify changes in the magnitude of the arm’s endpoint stiffness, the spatial information of the endpoint stiffness is lost. The grasp force is unsuitable for measuring a change in the orientation of the stiffness ellipse, which might be useful when examining how humans optimize their endpoint stiffness to counter unstable or unpredictable environments


Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive and lethal disease, lacking effective therapeutic approaches. Available therapies only marginally prolong patient survival and are frequently coupled with severe adverse events. It is therefore pivotal to investigate novel and safe pharmacological approaches. We have recently identified the ABC transporter, ABCC3, whose expression is dependent on mutation of TP53, as a novel target in PDAC. ABCC3-mediated regulation of PDAC cell proliferation and tumour growth in vivo was demonstrated and was shown to be conferred by upregulation of STAT3 signalling and regulation of apoptosis.To verify the potential of ABCC3 as a pharmacological target, a small molecule inhibitor of ABCC3, referred to here as MCI-715, was designed. In vitro assays were performed to assess the effects of ABCC3 inhibition on anchorage-dependent and anchorage-independent PDAC cell growth. The impact of ABCC3 inhibition on specific signalling pathways was verified by Western blotting. The potential of targeting ABCC3 with MCI-715 to counteract PDAC progression was additionally tested in several animal models of PDAC, including xenograft mouse models and transgenic mouse model of PDAC., remarkably slowing tumour growth in mice xenografts and patient-derived xenografts and increasing the survival rate in a transgenic mouse model. Furthermore, we show that stromal cells in pancreatic tumours, which actively participate in PDAC progression, are enriched for ABCC3, and that its inhibition may contribute to stroma reprogramming.Using both mouse models and human cell lines of PDAC, we show that the pharmacological inhibition of ABCC3 significantly decreased PDAC cell proliferation and clonal expansion in vitro and in vivoOur results indicate that ABCC3 inhibition with MCI-715 demonstrated strong antitumor activity and is well tolerated, which leads us to conclude that ABCC3 inhibition is a novel and promising therapeutic strategy for a considerable cohort of patients with pancreatic cancer.The online version of this article (10.1186/s13046-019-1308-7) contains supplementary material, which is available to authorized users. Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive malignancy with a dismal prognosis. In recent years, the incidence and mortality of PDAC is increasing, making PDAC the fourth most common cancer-related cause of death in the western world . Five-yeWe have recently demonstrated that the ABC transporter ABCC3  is an important regulator of PDAC signalling and that ABCC3 overexpression is a negative prognostic factor for PDAC patients . We showNo studies have so far investigated the pharmacological targeting of ABCC3 beyond the reversal of chemoresistance. In this study, we explore the potential of ABCC3 inhibition to counteract PDAC progression. From a long-running medicinal chemistry/drug discovery process, we identified a novel small-molecule inhibitor of ABCC3, which reduces PDAC progression in vitro and tumour growth and disease progression in vivo in several animal models. Importantly, we show that ABCC3 inhibition not only affects PDAC tumours, but also targets PDAC stroma, which is enriched in ABCC3. Collectively, the results suggest that targeting ABCC3 with a small molecule inhibitor such as MCI-715 provides an effective therapeutic approach for a cohort of PDAC patients that overexpress ABCC3.Mycoplasma-free conditions: AsPC1 (CRL-1682™), HPAFII (CRL-1997™), CFPAC-1 (CRL-1918™) and BJ (CRL-2522™). Human Immortalized Pancreatic CAF-stellate cells (CAFs) were purchased from Neuromics . All cell lines were cultured less than 3 months after resuscitation and regularly tested for Mycoplasma. Primary cell line (KPC) was established from pancreatic tumours of KRASWT/G12D, P53WT/R172H, PDX-1CRE+/+ (KPC) transgenic mouse model as described elsewhere [Cell lines were purchased from ATCC  and cultured as per manufacturer’s instructions in PC1 CRL-182™, HPAFThe role of ABCC3 in PDAC was assessed by transient knockdown with the use of siRNA transfection. For transient ABCC3 knockdown two siRNA sequences (siABCC3–1 (Hs_ABCC3_6), siABCC3–2 (Hs_ABCC3_15) QIAGEN) and control siRNA (siSCR) were used at working concentration of 75 nM. DarmaFECT 1 transfection reagent (Dharmacon) was used for the experiments according to manufacturer’s instructions. Western blotting was used to verify knockdown efficiency.4 cells/well in 12-well or 2 × 104 cells/well in 24-well cell culture plates, were treated in duplicate, with DMSO used as a negative control. After 72 h, viable cells were counted with trypan blue exclusion.The effect of ABCC3 inhibition with MCI-715 on anchorage-dependent growth was assessed by trypan blue exclusion assay. Cells, seeded at a density of 5 × 10To validate the effect of MCI-715 on tumorigenic potential of cancer cells and their ability to form colonies in anchorage-independent conditions, soft agar colony formation assay was performed as previously described , 9. ColoThe evaluation of protein expression was performed by Western blot analysis. Cells and tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with Protease/Phosphatase Inhibitor Cocktail  and sonicated. Proteins were separated by SDS-PAGE and detected by Western blotting according to standard procedures using the following antibodies (1:1000 in BSA 3%/TBST): ABCC3 , HIF1α , pSTAT3 Tyr705 , GAPDH , β-actin , ⍺-actinin , ⍺/β tubulin , cleaved caspase 3 , vimentin , α-SMA . Immunoblots were quantified using ImageJ and Image Lab 5.2.1 on the basis of three separate experiments.To verify the efficiency of ABCC3 blocking by sulindac and MCI-715, Calcein transport with and without ABCC3 inhibition was tested. Wild type ABCC3 cDNA encoded by recombinant pcDNA3.1 plasmid (pcDNA3-ABCC3) was a kind gift from Prof Susan Cole . pDsRed25 cells were seeded onto a T25 tissue culture flask and double transfected 24 h later with a transfection mix prepared from 7.5 μg pcDNA3-ABCC3 and 2.5 μg pDsRed in a 20 μl volume of 5% glucose and 17 μg of linear 25 kDa PEI . The DNA/PEI complex was diluted in 5 ml DMEM and added to the cells. After a further 24 h the culture was supplemented with butyric acid to a final concentration of 2 mM to stimulate transcription. The cells were harvested after a further 24 h in versene and aliquots (2 × 105 cells in 200 μl growth medium) and incubated with Calcein-AM  with 0 μM to 750 μM inhibitor (Sulindac or MCI-715), for 20 min at 37 °C. Stock solutions of Sulindac and MCI-715 were prepared in DMSO; the vehicle had no effect on the transport assay (data not shown). The cells were then washed twice by pelleting at 160 x G and resuspended in 0.5 ml ice-cold DMEM minus phenyl red but supplemented with FCS to 1% v/v. The cells were analysed using a FACScan flow cytometer . The population was gated for 10,000 single cells of normal size and granularity. Calcein content was measured in the FL-1 (green) channel, and red fluorescence from the expressed DsRed was measured in the FL-2 channel. Flow cytometry data were acquired using CellQuest Pro Software  and analysed using FlowJo . ABCC3 transport activity was inferred from the fold difference in Calcein content of untransfected cells versus the transfected cells. To compare independent datasets the fold difference was normalised to 100% activity in the absence of inhibitor. Statistical analysis of the dose response from three biological replicate experiments was by GraphPad PRISM® V5.0 software with IC50 determined by non-linear regression analyses .HEK293T cells were cultured as adherent monolayers in Dubecco’s Modifed Eagle Medium (DMEM) High Glucose  supplemented with 10% foetal calf serum (FCS). Transient transfection used polyethylenimine (PEI), as described previously . BrieflyPotential induction of apoptosis in PDAC cell lines after ABCC3 inhibition was verified by analysis of caspase 3/7 activity. PDAC cells were seeded in a 96-well cell culture plate at the density of 10.000 cells per well. The following day cells were treated with 10 μM MCI-715 and incubated with Incucyte Caspase 3/7 fluorogenic apoptosis detection reagent (1:1000) (Essen Bioscience) according to manufacturer’s instruction and monitored for up to 72 h using IncuCyte Life Cell Analysis Imaging System (Sartorius). The ratio between the increase in caspase 3/7 activity and cell confluence was calculated to assess the induction of apoptosis. Each experiment was performed in three biologically independent replicates.Cyclooxygenase activity was determined using the COX Fluorescent Inhibitor Screening Assay Kit  according to the manufacturer’s recommendation. Recombinant COX-1 or COX-2 were incubated with MCI-715 for 20 min prior to the addition of arachidonic acid before initiating the assays. The fluorescent readout from the assay was measured using a Biotek Synergy H4 plate reader. Sulindac sulfide served as a positive control for the assay.All animal experiments were performed according to standard national and institutional guidelines. The Curtin University Animal Ethics Committee approved procedures on KPC mice (AEC_2016_40). Xenograft studies were approved by the Italian Ministry of Health (N.484/2016-PR). All animals were kept at 21 °C in ventilated cages cleaned weekly, with 12 h light/ 12 h dark cycle and provided with water and food ad libitum. Sample size was estimated on the basis of the power calculations performed previously in our group .6 HPAFII cells in PBS (200 μl). When tumours reached a volume of 100 mm3 mice were randomized in groups of six animals and treated via oral gavage with 25 mg/kg of MCI-715 or vehicle (0.5% CMC/0.25% tween-80) three times a week, for three weeks. Mice were sacrificed when tumour volumes reached 1500 mm3. Patient-derived xenografts (PDXs) were established by engrafting samples of primary pancreatic cancer from patients after surgical resection into the right flank of 4–6-week-old female CD-1 nude mice. PDXs were passaged twice (P1 and P2) by sequential reimplantation in CD-1 mice. When tumours reached a volume of 50 mm3, animals bearing P2 PDXs were randomized into two groups (n = 6) and treated via oral gavage with 25 mg/kg of MCI-715 or vehicle three times a week, for three weeks and sacrificed as tumours reached 1000 mm3. Tumours were measured using a surgical calliper and volumes calculated according to the formula: tumour volume = (length * width2)/2.Female athymic CD-1 nu/nu mice (5–7 weeks old) from Charles River Laboratories  were injected subcutaneously in the right flank with 3 × 10WT/G12D, P53WT/R172H, PDX-1CRE+/+ (KPC) transgenic mice and control mice were maintained and genotyped by the Animal Research Centre  according to the original protocol [n = 8) daily by oral gavage. Animals were monitored daily and sacrificed when visible signs of pain and distress could be observed, such as significant weight loss , dehydration, development of ascites and breathing problems caused by developing lymphoma or pain. Mice were sacrificed by snipping of the main cardiac vein followed by organ perfusion through the heart. Survival of mice was plotted using a Kaplan-Meier curve and quantified using a log rank test.KRAS, are reflected in vivo, immunohistochemistry (IHC) analysis of murine tissues was performed.To verify whether the effects of ABCC3 inhibition on the signalling pathways, which activity was shown to be downregulated in vitroFormalin fixed-Paraffin embedded (FFPE) pancreas and liver tissues were cut into 4 μm sections. A pathologist blinded to the experimental groups assessed histopathological analysis by Haematoxylin and Eosin (H&E) staining. IHC staining followed standard protocols after heat-induced antigen retrieval with pH 9.0 Tris/EDTA unless stated otherwise ABCC3 , pSTAT3 Y705 , HIF1α , Vimentin . Apoptosis was assessed using ApopTag® Plus Peroxidase in Situ Apoptosis Detection Kit based on the TUNEL method (Millipore). Web-based software ImmunoRatio was used for quantitative analysis. For each treatment arm, at least 15 photos were quantified for statistical analysis.t-test (Western blot and IHC quantification), multiple t-test (tumour growth) and one-way ANOVA (cell growth) assuming independent samples and normal distributions were used. A 95% confidence interval was used for statistics and P < 0.05 was considered significant. Survival rates were described by a Kaplan-Meier curve and quantified by log rank (Mantel-Cox) test. Results are representative of at least three independent experiments and presented as mean ± SEM.Sample size for each experiment was assessed based on previous work . StatistWe have recently demonstrated that ABCC3 plays a prominent role in stimulating PDAC cell and tumour growth, suggesting the potential of ABCC3 as a novel target in PDAC therapy . We ther50 for MCI-715 was determined and found to inhibit ABCC3 with 2-fold higher potency than the parent compound sulindac sulfide    that resulted in an increased survival (not shown) was observed in PDX mice treated with MCI-715  were treated with 25 mg/kg MCI-715 by oral gavage and the tumour volume was measured every three days. A statistically significant reduction in tumour growth was observed in MCI-715 treated HPAFII xenografts compared to the vehicle treated group p = 0.043 , closely mimicking human disease [n = 8) and MCI-715 (n = 6) treatment groups. Mice were treated daily with 25 mg/kg MCI-715 by oral gavage. Strikingly, a statistically significant increase in survival was achieved with MCI-715 treatment compared to vehicle-treated mice (p = 0.0010),  were analysed. Western blot analysis of CAFs demonstrated increased ABCC3 expression in stromal cells compared to normal fibroblasts (BJ) Fig. b. Additin = 4) versus vehicle (n = 3)  Fig. e. SimilaIn our latest paper we have discussed the involvement of ABCC3 in the regulation of PDAC progression . ABCC3 aHere we have explored the potential of ABCC3 as a novel druggable target in PDAC. Due to the lack of effective and specific inhibitors of ABCC3, we investigated possible molecules with ABCC3-inhibitory activity. Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) showing anticancer activities that may involve mechanisms unrelated to its cyclooxygenase inhibitory activity, including the blocking of several ABC transporters . HoweverAlthough our data indicate the activity of MCI-715 towards ABCC3, other ABCC transporters, especially ABCC1, share a similar structure and may exhibit redundant functions to ABCC3, suggesting a possible cross-reactivity of MCI-715 with other ABCC transporters. In fact, as sulindac is known to have activity against ABCC1, it is reasonable to hypothesise that sulindac derivatives, as MCI-715 may also target ABCC1 , with otIn conclusion, we provide evidence of the pharmacological potential of ABCC3 inhibition with a small molecule inhibitor, MCI-715, to block PDAC progression in vivo without discernible toxicity. The clinical relevance of our results is emphasized by the fact that efficacy was demonstrated in both patient derived xenografts as well as KPC mice, a transgenic mouse model of PDAC that is the most predictive for pre-clinical evaluation of anti-tumorigenic agents for PDAC. In addition, sulindac, the scaffold on which MCI-715 was developed, is already approved for medical use, suggesting the possibility of rapid development down the translational path. Moreover, the oral bioavailability of MCI-715 presents an opportunity for a non-invasive and tolerable therapeutic approach. Overall, our findings support further preclinical validation and development of MCI-715 and related analogues for the treatment of pancreatic cancer.Additional file 1:Figure S1. MCI-715- a specific inhibitor of ABCC3. Figure S2. Pharmacological inhibition of ABCC3 with MCI-715 reduces the activity of pSTAT3 Y705 and HIF1α. Figure S3. MCI-715 treatment in the animal models of PDAC. Figure S4. MCI-715 treatment induces apoptosis in the KPC primary cell line. Figure S5. Modulation of ABCC3 activity influences the viability of epithelial and stromal PDAC cells. (ZIP 4873 kb)


VOC) of the DSSC, and thereby the photoconversion efficiency of the cell. In particular, electrolytes containing 1.4 g bare and cross-linked starches showed ionic conductivities of σ = 1.61, 0.59, 0.38, and 0.35 S cm−1, and the corresponding DSSCs showed efficiencies of 1.2, 1.4, 0.93, and 1.11%, respectively.Dye-sensitized solar cells (DSSCs) have become a validated and economically credible competitor to the traditional solid-state junction photovoltaic devices. DSSCs based on biopolymer gel electrolyte systems offer the perspective of competitive conversion efficiencies with a very low-cost fabrication. In this paper, a new starch-based biopolymer gel electrolyte system is prepared by mixing lithium iodide and iodine with bare and citric acid cross-linked potato starches with glycerol as the plasticizing agent. The effect of the preparation methods on the starch cross-linking degree as well as the photoconversion efficiency of the resulting DSSC cells is carefully analyzed. Fourier transform spectroscopy, X-ray diffraction, and scanning electron microscopy were used to characterize the morphology and conformational changes of starch in the electrolytes. The conductivity of the biopolymer electrolytes was determined by electrochemical impedance spectroscopy. DSSC based on the starch-gel polymer electrolytes were characterized by photovoltaic measurements and electrochemical impedance spectroscopy. Results clearly show that the cross-linking increases the recombination resistance and open circuit voltage ( The energy crisis is the most important global issue that human society is currently facing. This problem is mainly related to three factors: increasing energy demand to support the economy, depletion of fossil fuels, and the greenhouse effect due to combustion of fossil fuels ,2. There2, ZnO, or SnO2, and a dye sensitizer together which constitute the photoanode, an electrolyte containing a redox couple, and a counter-electrode. The mechanism of DSSCs involves the photo-excitation of the sensitizer and the injection of a photo-excited electron into the conduction band of the semiconductor oxide, generating the oxidized form of the dye. The electron from the conduction band of the semiconductor oxide layer reaches the substrate and flows through the external circuit. Then, the ground state of the dye is regained by electron donation from the reduced state of the electrolyte, which in turn is regenerated by the reduction of the oxidized species of the electrolyte at the counter-electrode. DSSCs can be produced at reasonably low cost while yielding relatively decent efficiencies  in 2-propanol and sintering at 450 °C for 30 min in a tubular furnace, and this electrode was used as the counter electrode for the DSSC.The Platinum (Pt)-coated FTO plate was prepared by drop casting 5 × 10Starch was cross-linked by the following three different procedures, identified as A1, A2, and A3.A1: 5 g of starch was mixed with 100 mL of distilled water, 2 mL glycerol, and 0.5 g of citric acid at room temperature for 5 min. This suspension was transferred to a water bath at 90 °C for 30 min and agitated by a magnetic stirrer. After cooling, the solution was poured into a Petri dish and dried at 50 °C. After drying, the product was weighed and used for electrolyte preparation .2CO3 were mixed with 100 mL distilled water and stirred for 5 min. Five grams of starch was added to this solution and stirred on a hot plate at 90 °C. After 20 min, 2 mL of glycerol was added and stirred. The resulted homogeneous solution was poured into a Petri dish and dried. The dried product was used for the preparation of electrolyte . In. In23]. Tg) [Tg. It is reported that the introduction of cross-links deprives the segmental motion of polymers and increases Tg due to the reduction in the number of chain units that could be thermally activated. Tg is an indirect measure of cross-link density [Tg of gel electrolytes is shifted to higher temperature when compared with bare starch-based electrolyte due to the restricted segmental mobility of starch by cross-linking with citric acid [The molecular motions of polymer chains are inhibited at low temperatures and increase when thermal energy is applied. The temperature at which the physical change of crystalline to flexible state takes place is called the glass transition temperature (Tg) . The inf density . From Firic acid .From σ) of the prepared gel electrolytes are calculated from the measured bulk resistance (Rb) values obtained in electrochemical impendence spectroscopy , and “A” is the active area, which is taken as 1 cm2. The equivalent circuit used and the circuit elements value are shown in the −1. Further, the conductivity decreased on cross-linking compared to E3, possibly due to the lack of –OH groups to interact with the –OH groups of glycerol. Additionally, as mentioned in the + in the electrolyte medium containing Li+ [The efficiency of the DSSC composed of gel polymer electrolyte is highly influenced by the mobility of ions, and hence we measured the conductivity of electrolytes . The controscopy  by usingning Li+ .−2 (1.5 AM solar simulator) are shown in VOC), (ii) short circuit current (JSC), and (iii) fill factor (ff) of the cell. JSC mainly depends upon ionic mobility in the electrolyte, VOC depends upon the recombination resistance and electron lifetime of electrons in the conduction band of TiO2 [The photocurrent-voltage measurements of the fabricated DSSC composed of liquid, bare starch, and cross-linked starch under 85 mW cm of TiO2 , and ff JSC) values due to high ionic conductivity. Apart from the lower value of conductivity (σ), EC1 showed better photocurrent conversion efficiency and VOC compared to E3. This may be due to the reduction in charge recombination reaction in the presence of cross-linked starch. This is further confirmed by EIS and electron lifetime measurements in the following sections. From +) introduced in the form of Na2CO3 and NaOH in EC2 and EC3, respectively, during cross-linking significantly influenced the performance of the DSSC and resulted in lower JSC values, probably due to the formation of ionic clusters, leading to the decrease in the effective concentration of free charge carriers [+ cation along with Li+ in electrolyte decreased the relative quantum yield for electron injection (φinj) from LUMO of dye to the conduction band of TiO2. All of these factors may contribute to the lower performance of the DSSCs composed of EC2 and EC3 compared to the DSSC composed of EC1.In the case of EC2 and EC3, the presence of sodium ions (Nacarriers . The pre2/dye/electrolyte interface. The increment in the diameter of this semi-circle implies the higher charge transfer resistance (RCT) at the TiO2/dye/electrolyte interface. The last semi-circle at the high-frequency region corresponds to Warburg resistance for tri-iodide diffusion, ZW. From RCT increases with the increase in the concentration of starch. Further, RCT is higher for DSSC composed of EC1 and accounts for higher open-circuit voltage and efficiency of DSSC.The interfacial resistance and electronic transport process of the DSSC were studied by the electrochemical impedance spectroscopic (EIS) analysis. fmax) and used for the calculation of electron lifetime at the conduction band of TiO2 using Equation (2):τ” is the electron lifetime and “f” is the maximum frequency. The electron lifetime values are given in τ increases with the increase in the concentration of starch and is higher for DSSC composed of EC1, showing the reduction in back electron transfer by the addition of starch and by cross-linking [The Bode phase plots of DSSC composed of different electrolytes are shown in -linking .−1 and DSSC with 1.2% efficiency. Cross-linking improved the electron lifetime and recombination resistance of DSSC and the photoconversion efficiency to 1.4% with conductivity 0.59 S cm−1. Results clearly show additional potential for further cost reduction and simplification of the manufacturing of dye solar cells.A new starch-based biopolymer gel electrolyte is prepared for the application of a dye-sensitized solar cell. The effects of preparation methods, as well as the degree of cross-linking on the physical properties and photoconversion efficiency of the resulting DSSC cells, are carefully analyzed. FT-IR spectroscopy of the gel polymer electrolytes shows the conformational changes of starch during cross-linking. X-ray diffraction reveals that the cross-linking processes affect the quality and quantity of the crystalline nature of starch. Cross-linking in starch also changes its glass transition temperature, as proved by differential scanning calorimetry. DSSCs composed of prepared electrolytes are characterized by electrochemical and photovoltaic measurements. The newly prepared electrolyte shows an ionic conductivity of 1.61 S cm


With the increasing need for kidney transplantation in the paediatric population and changing donor demographics, children without a living donor option will potentially be offered an adult deceased donor transplant of marginal quality. Given the importance of long-term graft survival for paediatric recipients, consideration is now being given to kidneys from small paediatric donors (SPDs). There exist a lack of consensus and a reluctance amongst some centres in transplanting SPDs due to high surgical complication rates, graft loss and concerns regarding low nephron mass and long-term function. The aim of this review is to examine and present the evidence base regarding the transplantation of these organs. The literature in both the paediatric and adult renal transplant fields, as well as recent relevant conference proceedings, is reviewed. We discuss the surgical techniques, long-term graft function and rates of complications following transplantation of SPDs. We compare graft survival of SPDs to adult deceased donors and consider the use of small paediatric donors after circulatory death (DCD) organs. In conclusion, evidence is presented that may refute historically held paradigms regarding the transplantation of SPDs in paediatric recipients, thereby potentially allowing significant expansion of the donor pool. The implication of changing donor demographics is that many children in the UK and USA that do not have a living donor option will be offered an adult deceased donor transplant potentially of marginal quality, despite efforts to match with organs possessing the lowest Kidney Donor Profile Index (KDPI). Whilst paediatric Eurotransplant kidneys are prioritised for paediatric recipients, this has not historically been the case in the UK or the USA due to concerns regarding technical complications. However, given the importance of long-term graft survival for paediatric recipients and recent paradigm shifts within individual units, consideration is now being given to new sources of organs which might sustain excellent renal function with longer graft survival. Attention has therefore turned to the use of organs from small paediatric donors (SPD).In many countries, rates of living donor renal transplantation appear to have reached a plateau or have even started to fall. There were approximately 1050 living kidney donors each year from 2010 to 2017 in the UK, whilst in the USA there has been a steady decline from 6279 living kidney donors in 2010 to 5627 donors in 2016 . Furthermore, there was a graft thrombosis rate of 8.3% (n = 386) in organs from donors under the age of 5 years, compared to 3.2% (n = 1667) in donors over 10 years [There exist a lack of consensus and a reluctance amongst centres in transplanting SPD organs due to an increased risk of thrombosis of the donor vessels. For the purposes of this review, we have defined a SPD as a donor weighing less than 20 kg, though definitions vary in the literature. An average child of 20 kg is approximately 6 years old, with a renal length of approximately 8 cm. A univariate analysis by Singh et al. of NAPRTCS registry data showed significantly higher rates of SPD graft loss due to thrombosis in paediatric recipients under 2 years old compared to those over 12 years old  [p < 0.01). In a European registry analysis of 429 transplants from paediatric donors aged between 0 and 5 years into paediatric recipients, the authors sought to examine the association of donor-recipient age combination with 5-year graft survival [Studies examining outcomes following transplantation using SPDs (with more than 20 recipients) are shown in Table  < 0.01) . This wasurvival . PerformThese reports consistently demonstrate good long-term graft survival but with an increase in the risk of early graft loss from thrombosis. However, it is important to consider that a majority of these analyses are historical and there exist no recent large-scale reviews or randomised controlled trials. Furthermore, the quality of deceased donor organs and the use of anti-thrombotic protocols have evolved since this data was collected.p = 0.02) compared to the adult donor groups, with both SCD and ECD 5-year graft survival at 75%. However, a small review by Stey et al. demonstrated contrasting findings, with inferior outcomes in the SPD group [A number of investigators have sought to compare the outcomes of SPD kidney transplants to matched cohorts of adult standard criteria donor (SCD) transplants , 23, 28.PD group . The autDespite the historically high technical failure rate associated with transplantation of SPDs, it appears overall that long-term outcomes are superior when compared to matched cohorts of adult SCD grafts. The published outcomes are excellent even when compared with outcomes following living donor transplantation , 23. CleUnsurprisingly, there are currently no randomised controlled trials comparing outcomes following EBK transplantation to single kidney transplantation, in part due to the low frequency of such transplants at centres throughout the world. Comparison of the techniques is confounded by the fact that kidneys undergoing en bloc transplantation are typically smaller with a lower mass than those that may be considered for single implantation. Furthermore, damage to the en bloc specimen at retrieval may result in one organ being discarded and the other organ being transplanted singly. This will likely increase the risk of complications, reduce nephron mass and affect long-term survival.Studies which have attempted to compare EBK and single kidney transplantation are shown in Table p < 0.001). The previously described registry review by Bhayana et al. in 3198 SPD organ recipients demonstrated superior EBK transplantation outcomes at 1 and 10 years (85% and 64%) compared to single kidney transplantation (81% and 53%) at similar time points [. analysed 2449 recipients of SPD kidneys [3 vs 45 ± 12 mm3, p < 0.01); however, at 1-year post-transplantation, the graft sizes from both groups were similar .The strongest evidence that EBK transplantation might provide superior graft survival comes from several large US registry reviews –11, 15.  kidneys . Recipie kidneys . The autWhilst these results are encouraging, there still exist a paucity of strong evidence of the long-term outcomes and the feasibility of utilising kidneys from infant donors. Several investigators have suggested that there may be a lower age threshold, beyond which results are inferior , 33. HowThere is compelling evidence to suggest that overall EBK transplantation provides excellent graft survival and function. However, to maximise the utilisation of a scarce resource and extend access to the benefits of transplantation to as many recipients as possible, the question remains as to whether single kidney transplantation to two recipients is more appropriate. Laurence et al. constructed a decision analysis model to predict the outcome in life years for patients on the transplant waiting list depending on whether they received an EBK or single kidney transplant . The autTo expand the donor pool and decrease waiting times, there has been increased utilisation of SPD organs transplanted into adult recipients, with the very small organs transplanted en bloc. Although an increased risk of graft thrombosis and hyperfiltration injury has been demonstrated with the use of EBK transplants, reports in adult recipients show excellent outcomes. Currently, only a small percentage of renal transplantation in paediatric recipients utilise SPDs. Children undergoing renal transplantation pre-emptively or after a short waiting time have not only survival benefit but also better growth and cognitive development compared with children receiving dialysis. Therefore, expanding the donor pool in the paediatric population is of paramount importance to enable transplantation as soon as possible in this cohort.., outcomes of paediatric renal transplant recipients from donors less than 6 years old were reported and compared to those from older donors. Graft survival was comparable in both groups at 1 year (77 vs 76%) and 5 years (55 vs 60%) [The large US-based registry reviews previously described analyse long-term outcomes in combined adult and paediatric recipient groups; none however perform sub-group analysis to assess the outcomes of using SPD kidneys in paediatric recipients –12. Stud vs 60%) .Despite these promising results, there is evidence that suggests allograft function and survival are poorer when both recipient and donor are young. Singh et al. in a univariate analysis of 4394 transplants, showed that graft loss due to thrombosis was significantly higher in children less than 2 years of age compared to that in older groups (9% vs 3.5% when recipients are older than 12) . RecipieIt appears that the risk of transplantation with very small SPD kidneys in both children and adult recipients is greatest in the early postoperative period. However, it is clear that children receiving organs from older paediatric donors have comparable or superior long-term functional outcomes compared to those that receive adult allografts.The use of DCD organs in adults has become an accepted way of recovering more organs for transplantation and has allowed expansion of the donor pool. However, increasing acceptance and utilisation of paediatric DCD organs remain challenging for a variety of reasons. There exist limited experience and comfort with transplanting such organs and few standardised protocols for their use, due to concerns over compromised graft survival. There is also considerable ethical and emotional concerns surrounding the use of such organs.n = 405) cohort of paediatric DBD organ recipients [Workman et al.’s recent review of data from the Organ Procurement and Transplant Network (2001–2010) showed a 10% decrease in the use of paediatric deceased donor solid organs over the 10-year period . During cipients . The autIn contrast to similar studies examining DCD paediatric transplantation, Vries et al. identified that 12% of the DCD kidneys within their analysis were considered Maastricht category 2, defined as uncontrolled donation following unsuccessful resuscitation . The autDion et al. in a recent single-centre analysis identified 21 adult recipients of EBK transplants from paediatric donors under the age of 4 years old, of which four organ pairs were obtained from DCD donors . DGF ensAt present, therefore, there is minimal data regarding the use of small paediatric DCD organs in children. Whilst results obtained from adult DCD organs, when implanted into adults, are equivalent to those from DBD organs, a recent UK registry data analysis has demonstrated that adult DCD organs, when transplanted into children, possess outcomes that are comparable to DBD organs or organs from live donors . However. and Winnicki et al. both demonstrated marginally inferior outcomes in SPD organs at 1-year follow-up compared to adult SCD organs; however, this had equalised by follow-up at 5 years with comparable survival rates between organ types and techniques [p = 0.85), whilst the EBK transplantation group had significantly shorter wait times, between activation and transplantation, compared to that of the SCD group .Kidney transplantation is the treatment of choice for children with end stage renal disease. It is well established that patient survival, quality of life, growth and cognitive development are superior with transplantation compared to dialysis . As donochniques , 16. WinHistorically, there has been reluctance to transplant SPD organs in children, due to concerns of a higher incidence in surgical complications; however, the more recent published reviews have shown lower complication rates , 16. ThiNational and locally led strategies are needed to reduce paediatric transplant waiting times and increase the deceased donor pool by greater utilisation of SPD organs. Improved utilisation requires tackling several weaknesses in the organ transplant pathway. SPD organ procurement rates are low and whilst potential causal agents have been considered, specific rationales have not been analysed and documented , 36, 42.Another opportunity to increase transplantation rates would be to lower the threshold for en bloc splitting, to allow single kidney transplantation into two different recipients. Further evidence is required to determine the optimal donor weight for single-organ kidney and EBK transplantation; however, we would suggest a sensible minimum threshold of 15 kg when considering single kidney transplantation from SPDs. The decision to transplant organs from SPDs and the choice of performing EBK versus single kidney transplantation require careful consideration and assessment to ascertain that maximal benefit is attained for individual recipients and the remaining patients on the waiting list. When considering splitting, the surgeon should also consider the surgical approach and potential space required in the recipient. EBK transplantation into adult recipients within the extraperitoneal iliac fossa is well recognised and commonly performed. A similar approach may also be suitable in older children and adolescents; however, smaller children are likely to need an intra-peritoneal approach. In all but the smallest children, there is adequate space to transplant small EBKs within the abdominal cavity without developing abdominal compartment syndrome. However, there are no models described within the literatures to determine the available space or to stratify the risk of compartment syndrome; the authors would therefore recommend a case by case assessment by an experienced surgeon.There is no doubt that centre volume and surgeon experience will affect graft survival rates, especially in very small paediatric recipients. Centralising specialist services in high-volume centres will result in better patient and graft outcomes. In the UK, two NHS hospital trusts  have been appointed as national specialist leads for utilisation of organs from donors below 2 years old. Discussions are ongoing in the UK about the provision of expertise for such retrievals. Clearly, evidence will be required that any additional resources result in a significant benefit.In conclusion, many SPD kidneys are excellent quality organs and have the potential to significantly expand the paediatric donor pool. These organs will allow the benefits of transplantation to be extended to many children and provide good long-term graft function, but carry an increased risk of perioperative thrombosis. It is likely that en bloc organs are to be preferred for small donors, although the cut-off point is unclear. Similarly, the minimum donor weight is uncertain, although organs from neonates have been transplanted. Using organs from small paediatric donors for implantation into children is feasible, and probably offers at least equivalent outcomes to ECD donors, but organs from very small donors should probably not be used widely until further data is available. We advocate a careful but considered approach to the use of these organs. We encourage stratagems to increase retrieval and utilisation rates as well as techniques to improve long term-outcomes from transplantation of SPD organs.


We developed a novel team-based Translational Journal Club utilizing three-member teams to find a basic science paper and two clinical study papers that covered a single therapeutic, either a pivotal study or a dissemination and implementation study; one member of the team presented a paper on the above topics in successive weeks. In addition, the trainees attended lectures on: how to design a pivotal clinical trial, dissemination and implementation, and drug development from a basic science discovery through its approval. From these presentations, the trainees appreciated the T0 to T3/4 continuum and its challenges. They also attended sessions on how to present scientific concepts, making them better communicators. The trainees found the Translational Journal club to be very rewarding, illuminating, and providing a much better understanding of the translational research processes required to develop new therapies.The mission of the National Center for Advancing Translational Science (NCATS) is to catalyze the generation of innovative methods and technologies that will enhance the development, testing, and implementation of diagnostics and therapeutics across a wide range of human diseases and conditions. Journal clubs organized by either departments or training grants are commonplace in academic medical centers. Most of these journal clubs focus on a single paper per session, and the topic is often related to the focus of the training grant, mentor, or the department and usually goes into great detail to dissect the strengths and weaknesses of the study design. The format of these journal clubs is fairly standard and explores the paper section by section with a critique and discussion of each section with an overall wrap up. While the importance of the paper may be discussed, it is usually within a very narrow focus and does not span consideration of the translation of the discovery all the way through to a therapeutic agent. Rarely is there a succession of presentations with a single theme.1 NCATS funds T32 training grants through the Clinical and Translational Science Award Program titled TL1 Clinical Research Training Awards. Our institution is the recipient of one of these grants focused on training predoctoral trainees. The grant funds 10 trainees yearly, including both PhD and MD/PhD trainees in their graduate years. Given the mission of NCATS, we decided to develop a novel Translational Journal Club that would educate TL1 trainees on the process of translation from discovery to dissemination and implementation. Since another focus of NCATS is the promotion of team science, our approach would also promulgate a team-based approach. The trainees were divided into groups of three and tasked with working as a team to choose three papers that would exemplify translational research from T0 to T3/4. Thus, the Translational Journal Club focused on the translation of fundamental discoveries and also working as a team.The mission of the National Center for Advancing Translational Science (NCATS) is to catalyze the generation of innovative methods and technologies that will enhance the development, testing, and implementation of diagnostics and therapeutics across a wide range of human diseases and conditions.The schedule for the Translational Journal Club is outlined in Table One member of the team presented a publication in successive weeks: 1) a basic science publication that ultimately led to a clinical trial, (2) a corresponding clinical trial publication, and (3) a large-scale clinical trial publication or a publication that provided guidance on the use of the therapeutic clinically (dissemination and implementation). All trainees were encouraged to participate in discussion about the presentations. After all of the team’s presentations, the trainees were given a series of questions pertaining to the three papers and they had 1 week to answer them . In addition, four open-ended items were included to collect more detailed information on: (1) How the journal club differed from the student’s prior journal club experiences?; (2) The student’s primary reason for scoring the NPS “likelihood to recommend” question; (3) The team process for selecting papers; and (4) How the journal club can be improved for next year?. The study was classified as program evaluation/quality improvement by the Institutional Review Board.To assess the impact of the Translational Journal Club and the perceived value of participation, the trainees were given an electronic survey to complete Table  at the eOur goal was not only to enhance the trainees’ understanding of the processes from T0 to T3/4, but also to challenge them to work outside of their comfort zones. At the same time, we wanted the trainees to appreciate the power of working as a team to explore an area for which they were not familiar and learn from each other. They collaborated on topic identification and paper selection. The trainees chose the topic based on several different rationales. For example, one team chose papers associated with Rituximab because one of the members saw a patient receiving the drug in a clinic that she attended as a requirement for the TL1 program. In another situation, the papers were chosen because of interest in the disease (hepatitis C) due to familiarity with a patient with the disease. A third was chosen for the novelty of the therapeutic approach (siRNA). The trainees often supplemented their presentations with information from sources other than the publications, demonstrating that they embraced the idea of developing an in-depth understanding of both the data in the paper and additional background information. This was a significant factor for the presentations since the trainees had, at best, limited prior knowledge concerning the basic science that ultimately led to the therapeutic. Similarly, they did not have significant clinical knowledge but were able to present the clinical papers in a cogent manner.To ensure that the trainees comprehended the concepts behind the papers, they were given a series of five questions to answer after each team’s presentation of the three papers. The trainees were able to provide cogent answers to defend their choice of a yes or no answer for each question. However, as noted in the survey Table  . The trainees all stated that this was a very unique journal club format and they had not experienced a similar format before. Some descriptions of the Translational Journal Club were, “fun, truly enjoyed attending, looked forward to attending, engaging, entertaining, informative and provided a unique opportunity to interact with peers.” The trainees stated that they liked the continuity of the presentations and the discussions; in particular, getting the “big picture” was an important component. They also liked not dissecting the paper as is often done in the typical journal clubs, although there were critiques and discussions. They all gained more appreciation for how a basic science discovery proceeds through clinical trials  and ultimately becomes a drug, and the time that it takes to develop a new therapeutic. Interestingly, there were no negative comments. There were constructive recommendations about including a clinician’s perspective, exploring topics outside of drug discovery, and revising the final questionnaire.In addition to the survey, the program director and associate program director met with the trainees at the end of the semester to solicit their thoughts on the Translational Journal Club. A presentation that the trainees found most interesting was the development of the therapeutics for cystic fibrosis, given by one of our clinical faculty whose expertise is in cystic fibrosis and who has participated in many clinical trials. Seeing the development of a basic science discovery into a new drug used in the clinic was most illuminating for them; in particular, they noted the length of time that it took from a fundamental discovery to an approved drug. A consensus suggestion was for a clinician with expertise in the disease area being discussed to join the discussion, so that the trainees could get a better appreciation of the disease that was being treated and the established therapeutic approaches. They also found it interesting to discover how practicing physicians get information about new drugs. A topic suggested for future journal clubs to further enhance their understanding of the T0–T3/T4 process was a lecture about the FDA approval process for new drugs.3 described the “characteristics of successful journal clubs included regular and anticipated meetings, mandatory attendance, clear long- and short-term purpose, appropriate meeting timing and incentives, a trained journal club leader to choose papers and lead discussion, circulating papers prior to the meeting, using the internet for wider dissemination and data storage, using established critical appraisal processes and summarizing journal club findings.” Our Translational Journal Club implemented many of those characteristics. One significant difference was that we used the team approach for choosing the papers rather than a trained journal club leader. In this regard, we feel that the trainees benefited greatly from the Translational Journal Club because they had to demonstrate that the chosen papers would stand up to a critical/rigorous assessment and questions from classmates, the program directors, and/or mentors. We used a questionnaire at the end of the team presentations to assess the summarization and comprehension of the three papers.This paper describes a team-based translational science approach journal club that to our knowledge is unique and previously unreported. Deenadayalan et al.4 described a journal club that created a liaison between practicing physical therapy clinicians and academic physicians to impart an evidence-based approach to physical therapy procedures. It utilized a three-step approach of evaluating randomized clinical trials, systematic reviews, diagnostic papers, and clinical guidelines. It appeared that most of the discussions were led by the academic physicians. Another journal club focused on a multidisciplinary approach to burn therapies.5We conducted a Pubmed search looking for descriptions of journal clubs that utilized a similar approach. We found several that had an implementation-based approach but none that emphasized a T0–T3/4 approach. One paperOverall, the Translational Journal Club was viewed positively by the trainees. They noted gaining a greater appreciation of translational research, the challenges associated with it, and the significant length of time it takes from discovery to approval and implementation of a new therapeutic. The trainees also learned to work together as a team to choose the papers for discussion. Based on the feedback that we received from the trainees, one addition to be made in future years is to enlist participation of a clinician to be part of the team.In summary, we have devised a novel team-based Translational Research Journal Club that can be easily implemented by other training programs or departments.


Concerns have been raised that the transplants performed in the local context at the time of procedures reported in this article  may haveDetails as to the donor sources and methods of obtaining informed consent from donors were not reported in , and theIn addition, the authors did not provide documentation when requested by the journal to confirm that the study had institutional ethics approval, and they did not respond to inquiries about the availability of underlying data supporting this study.PLOS ONE Editors retract this article.Owing to the lack of documentation to demonstrate this study had prospective ethical approval, insufficient reporting, unresolved concerns around the source of transplanted organs and whether they included organs from prisoners, and in compliance with international ethical standards for organ/tissue donation and transplantation, the The authors did not respond to the retraction notification.


ModelTest-NG is one to two orders of magnitude faster than jModelTest and ProtTest but equally accurate and introduces several new features, such as ascertainment bias correction, mixture, and free-rate models, or the automatic processing of single partitions. ModelTest-NG is available under a GNU GPL3 license at  It is well known that the use of distinct probabilistic models of evolution can change the outcome of phylogenetic analyses . Not surData and models supported: ModelTest-NG supports both nucleotide and amino acid models. It uses statistical criteria for selecting the best-fit substitution models such as AIC Partitioned MSAs: ModelTest-NG can automatically perform model selection on single, nonoverlapping partitions, as specified by the user .Phylogenetic templates: Users can select so-called templates for popular phylogenetic inference tools like RAxML  . This library encapsulates all compute- and memory-intensive phylogenetic likelihood computations and fully leverages the capabilities of modern x86 processors by using the AVX and AVX2 vector instruction sets. PLL also incorporates a recent algorithmic technique for accelerating likelihood calculations .ulations . All reqParallel execution: ModelTest-NG can take advantage of multicore desktop computers and clusters using PThreads and MPI (Message Passing Interface)  using simulated as well as empirical data sets. We measured model selection accuracy  using the simulated data sets, as well as run times. In all cases, we used the default model selection parameter settings. The experimental setup is described in detail in the ModelTest-NG found the true generating model for 81% of the simulated DNA MSAs  and for 85% of the simulated protein MSAs  . In geneIn terms of speed, on simulated DNA data, ModelTest-NG was 110.77 times faster than jModelTest but slower than ModelFinder (the latter was 1.59 times faster). On empirical DNA data, ModelTest-NG yielded average speedups of 510.13 over jModelTest and of 1.24 over ModelFinder  online. To ensure a fairer comparison with SMS, which only considers a subset of the models, we conducted a separate set of experiments comparing only ModelTest-NG and SMS on empirical data. For DNA data, both tools selected the same model 80% of the time, while ModelTest-NG was 95.53 times faster. For protein data, ModelTest-NG selected the same model as SMS 86.7% of the time and was 17.20 times faster.The thoroughness of the model parameter optimization routines in ModelTest-NG can be controlled by the user. In additional experiments, we found that the more thoroughly we optimize the likelihood score the more accurate the selected model becomes see  online. ModelTest-NG represents a substantial improvement over our previous tools, jModelTest and ProtTest. Although being equally accurate, it is up to two orders of magnitude faster on empirical data. Compared with ModelFinder, we observed similar run times for empirical data sets, but ModelFinder was faster on synthetic MSAs, particularly on DNA data. However, the accuracy of ModelFinder on DNA data was substantially lower than for ModelTest-NG . In future versions of ModelTest-NG, we intend to introduce new methods to dynamically determine the optimal speed/accuracy tradeoff for the data set at hand. ModelTest-NG is particularly well suited for analyzing large data sets.msz189_Supplementary_DataClick here for additional data file.


Maize starch plays a critical role in food processing and industrial application. The pasting properties, the most important starch characteristics, have enormous influence on fabrication property, flavor characteristics, storage, cooking, and baking. Understanding the genetic basis of starch pasting properties will be beneficial for manipulation of starch properties for a given purpose. Genome-wide association studies (GWAS) are becoming a powerful tool for dissecting the complex traits. Here, we carried out GWAS for seven pasting properties of maize starch with a panel of 230 inbred lines and 145,232 SNPs using one single-locus method, genome-wide efficient mixed model association (GEMMA), and three multi-locus methods, FASTmrEMMA, FarmCPU, and LASSO. We totally identified 60 quantitative trait nucleotides (QTNs) for starch pasting properties with these four GWAS methods. FASTmrEMMA detected the most QTNs (29), followed by FarmCPU (19) and LASSO (12), GEMMA detected the least QTNs (7). Of these QTNs, seven QTNs were identified by more than one method simultaneously. We further investigated locations of these significantly associated QTNs for possible candidate genes. These candidate genes and significant QTNs provide the guidance for further understanding of molecular mechanisms of starch pasting properties. We also compared the statistical powers and Type I errors of the four GWAS methods using Monte Carlo simulations. The results suggest that the multi-locus method is more powerful than the single-locus method and a combination of these multi-locus methods could help improve the detection power of GWAS. Zea mays L.) is the world’s most important crop for food, feed and industrial materials. Starch is the principal constituent of maize kernels, which accounts for approximately 70% of the kernel weight (Maize (l weight . Benefitl weight . Recentll weight . HoweverGenome-wide association studies (GWAS) provide the opportunity to decipher genetic architectures of complex traits in crops . Owing tSSIIa of maize affected the starch structure and physiochemical properties. ae1 and sh2 were associated with starch pasting properties. ZmBT1 gene with seven pasting properties. Zmisa2 underlying pasting properties of maize starch and proposed that these markers may be potentially utilized for marker-assisted selection. However, all of the above studies were based on specific candidate genes involved in kernel starch biosynthesis. Therefore, more comprehensive studies are required to further understand the molecular mechanisms of starch pasting properties. To our knowledge, GWAS for pasting properties of maize starch have not been reported up to now.There have been a few studies focusing on the genetic basis of pasting properties in maize starch. In this study, a worldwide collection of 230 inbred lines were genotyped with 145,232 SNPs using genotyping-by-sequencing (GBS) technology. Starch pasting properties including peak viscosity (PV), trough viscosity (TV), final viscosity (FV), breakdown viscosity (BD), setback viscosity (SB), pasting time (PT), and PTP were measured for the 230 lines using the Rapid Visco Analyser (RVA). The main objectives of this study were to (i) identify loci that are significantly associated with pasting properties of maize starch using single-locus and multi-locus GWAS methods, and (ii) compare three multi-locus methods  with one single-locus method (GEMMA) in terms of their detection powers and Type I errors.In this study, an association panel of 230 maize lines collected from the tropical, subtropical or temperate zone, representing a wide range of diversity, was used for GWAS. All the materials were planted with a randomized block design of three repetitions in the field of Sanya, Hainan province. At the four-leaf stage, young leave tissues were collected from each line and preserved at −80°C. DNA was extracted from the freeze-dried leave tissues with the modified CTAB method . After hThe panel of 230 maize inbred lines was genotyped using a GBS strategy. The ApeK1 restriction enzyme was used for library preparation, and GBS was performed on an Illumina platform by Novogene Bioinformatics Institute, Beijing, China. After quality control, a total of 145,232 high-quality SNPs with minor allele frequency (MAF) above 2% and missing rate below 20% remained to perform GWAS.The pasting properties of maize starch were evaluated using RVA . Three grams of starch obtained from each line was mixed with 25 ml of distilled water in the RVA canister. The RVA profile took a heat–hold–cool temperature cycle as follows: (1) set at 50°C as the starting temperature and maintained for 1 min; (2) heated to 95°C and held at 95°C for 2.5 min; and (3) cooled to 50°C and kept at 50°C for 1.4 min. The total processing time was about 12 min. The pasting properties were determined using a fixed paddle rotation at the speed of 160 r/m. The RVA parameters were recorded in centipose (cP). The pasting parameters obtained from the pasting curve including PV, TV, FV, PTP, PT and their derived parameters, BD and SB were recorded for all the inbred lines. The average value of three biological replicates from each line was obtained for data analysis.1. Principle component analysis (PCA) was used to control for population structure. Both single-locus and multi-locus methods were used to identify significant QTNs for seven starch properties. GEMMA was used for single-locus GWAS, and FASTmrEMMA  for GEMMA and FarmCPU. To mine candidate genes based on the detected QTNs for the pasting properties, we used gene annotation and ontology information available in maizeGDB2 and Phytozome database3.In this study, GWAS were performed in the association panel composed of 230 diverse maize inbred lines with 145,232 high-quality SNPs. The decay distance of LD across the whole genome was determined by software PopLDdecaySTmrEMMA , LASSO  analysis (Table 2). These results indicate that the phenotypic variations of starch pasting properties are mainly affected by genetic factors, and therefore this panel can be used for further genetic analyses. To determine the correlation among the seven pasting properties, the Pearson’s correlation coefficients were calculated. The results of the correlation analysis are illustrated in Figure 1. All the pairwise correlations between any two pasting properties exhibit significantly positive or negative correlations except three correlations between PT and TV, between PT and SB, and between SB and PTP.The descriptive statistics of the seven pasting properties for the 230 maize inbred lines are listed in analysis . The narFigure 2. According to the scree plot, the variance of principle component score decreases quickly until the fifth principle component . Therefore, we selected the first five principal components to control the population structure. All filtered SNPs were used to determine LD decay. A monotonic decrease in LD is found with increasing distance . At r2 = 0.2, the overall LD decay decreases dramatically to 10 kb. The genome-wide LD decay distance is about 250 kb at the cut-off of r2 = 0.1.In this study, PCA was used to correct for population structure. PCA plots of this association population are illustrated in Table 3. A total of 60 significant QTNs are identified for seven starch properties from the four GWAS methods. FASTmrEMMA detects the most QTNs (29), followed by FarmCPU (19) and LASSO (12), GEMMA detected the least QTNs (7). The numbers of significant QTNs detected for starch properties PV, TV, BD, FV, SB, PT, and PTP are 14, 10, 8, 12, 12, 6, and 5, respectively, from all the four methods. The corresponding numbers of the significant QTNs are 8, 6, 5, 3, 6, 1, and 4 from FASTmrEMMA; 2, 4, 2, 7, 4, 2, and 1 from FarmCPU; 2, 2, 0, 1, 3, 3, and 1 from LASSO; and 2, 0, 1, 1, 1, 2, and 0 from GEMMA. The largest QTN detected by FASTmrEMMA, FarmCPU, LASSO, and GEMMA explains 9.35, 14.96, 1.03, and 12.03(%) of the phenotypic variation, respectively. Among these significant QTNs, seven QTNs appear to control more than one trait (pleiotropic effect). For example, three QTNs  have significant effects on PV and TV. Both SNP_6_109456130 and SNP_7_160060597 are associated with PV and BD. The correlations between PV and TV as well as between PV and BD are significant.In this study, GWAS were conducted for 230 maize inbred lines with 145,232 SNPs using four methods and the results are listed in R2 values (proportion of phenotypic variance explained by the QTL) of the co-identified QTNs detected by different methods are completely different, whereas the signs of effects for these co-identified QTNs for the same trait are consistent. For example, the estimated effects of SNP_2_9506602 are −0.177, −0.057, and −0.244, and the corresponding R2 values are 5.14, 0.53, and 9.74(%) for trait PT when using FarmCPU, LASSO, and GEMMA, respectively. All the three methods demonstrate that this QTN has the negative effect on PT.When comparing the results across different methods, only seven common QTNs are identified by more than one method simultaneously. Among these QTNs, SNP_2_9506602 is detected across three GWAS methods ; SNP_9_103241537 and one pleiotropic QTN (SNP_4_89429269) are identified by FASTmrEMMA and FarmCPU simultaneously; SNP_4_144401228 is detected by FASTmrEMMA and LASSO; SNP_9_109684667 is detected by LASSO and GEMMA; SNP_3_12888452 is detected by FarmCPU and LASSO. SNP_7_173235732 associated with SB and FV are detected by FASTmrEMMA and FarmCPU, respectively. Note that the estimated effects and Table 4. The average powers for FASTmrEMMA, FarmCPU, LASSO, and GEMMA were 55.19, 43.31, 53.69, and 40.44(%), respectively, indicating the highest average power of FASTmrEMMA. However, different methods may be suitable for detection of different QTL. For example, FASTmrEMMA has the highest power for detecting QTL1, QTL4, QTL5, QTL6, and QTL8 but the lowest power for detecting QTL7. LASSO is the best method for detecting QTL2 and QTL3, whereas it is the worst method for detecting QTL1. GEMMA has the lowest power of detecting all the simulated QTL, but it is the most efficient method for detecting QTL7. Type I errors for all the four methods are also listed in Table 4. LASSO has the lowest Type I error, followed by GEMMA and FASTmrEMMA, and FarmCPU has the highest Type I error. The Type I errors of the four methods are under 0.0001 with the same order of magnitude. Overall, the Type I errors are well controlled for all the four approaches, and the three multi-locus approaches are more powerful than the single-locus approach.Simulation experiments were performed to compare the statistical powers and Type I errors of the four GWAS methods. The statistical powers of detecting the simulated QTL calculated based on 200 simulations are given in Arabidopsis thaliana, human and maize. In previous simulation studies, Bonferroni multiple test correction was used for single-locus method. However, it may be too strict to use Bonferroni correction (0.05/m) as the cut-off as not all loci are independent (m) for GEMMA. The results of simulation showed that Type I error of GEMMA with 1/m as the cut-off was well controlled and similar to that of three multi-locus methods. Additionally, the permutation method is commonly used to adjust for multiple tests, which yields reliable outcome but requires a lot of time for huge samples  encodes starch branching enzyme, which is a downstream gene involved in the final product of starch biosynthesis , which were classified into three main types containing biological process, molecular function, and cellular component . Under the first type, the most significant GO terms are gluconeogenesis process and hexose biosynthetic process, which play important roles in starch biosynthesis. Under the second type, these genes were significant related to chorismate synthase activity and glucose-6-phosphate isomerase activity. Under the third type, several genes were involved in photosystem. We also found that some candidate genes were involved in multiple functions. For example, GRMZM2G065083 are involved in gluconeogenesis process, hexose biosynthetic and metabolic process and glucose-6-phosphate isomerase activity. However, these genes were not found to be known genes involved in starch biosynthesis pathway, indicating that our study of the molecular mechanisms underlying pasting properties of maize starch is incomplete. These identified QTNs and candidate genes provide foundation for further functional studies to dissect the genetic mechanism manipulating maize pasting properties.Genome-wide association studies have been applied to dissect genetic architectures of several complex traits in maize . Howeverynthesis . It was e starch . The homd embryo . Severald embryo . To bettd embryo . The GO In this study, single-locus and multi-locus GWAS methods were used to identify loci associated with starch pasting properties in maize. A total of 60 significant QTNs were detected for seven pasting properties, of which 29, 19, 12, and 7 QTNs were detected using FASTmrEMMA, FarmCPU, LASSO, and GEMMA, respectively. These QTNs could be utilized for further genetic and breeding studies to regulate starch pasting properties. Additionally, we compared four GWAS methods for their detection powers and Type I errors based on simulation studies and found that the multi-locus method is more powerful than the single-locus method and the combination of these multi-locus methods could help improve the statistical power of current GWAS.CX and ZY designed the research plan. YX, TY, YZ, SY, PL, JL, and SX performed the experiments. YX and PL analyzed the data. YX wrote the paper. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The extruded UPFW showed a lower gelatinization degree than the extruded UPFP, which may be due to the higher non-starch polysaccharide content of the former. A high TDF content was recorded in both extrudates (12.4% in UPFP and 18.5% in UPFW), including a significant RS fraction. The water retention capacity and solubility indices were higher in the extruded flours, particularly in UPFW, while only marginal differences in oil retention capacity were observed among the products. The addition of UPFP or UPFW  to a yogurt did not alter the viscosity of the product, an important characteristic for the consumer's approval. Moreover, the composite yogurt showed a relatively low starch digestion rate. Extrusion of UPFs may be an alternative for the production of functional ingredients with important DF contents.There is a growing interest in the development of functional ingredients, including those with high indigestible carbohydrate content. Unripe plantain flour (UPF) is a source of indigestible carbohydrates, type II resistant starch (RS) in particular. A major drawback of UPF, however, is that its RS content decreases sharply after wet heat treatment. Here, we explore the possibility of preparing an extruded UPF-based functional ingredient that retains limited starch digestibility features and high dietary fiber content. Both an unripe plantain pulp flour (UPFP) and a whole (pulp and peel) unripe plantain flour (UPFW) were prepared, extruded under identical conditions and evaluated for their gelatinization degree, total starch (TS), resistant starch (RS), and total dietary fiber (TDF) content; functional properties, such as pasting profile, water retention capacity, and solubility, and oil absorption index were also analyzed. The extruded functional ingredient was added to a yogurt and the rheological characteristics and  In the last years, the end-use of plantain and other varieties of banana has increased around the world. Only in the present year, SCOPUS database shows 14 publications on the use of banana flour in foods, composite films, and modifications to improve its functional and physicochemical features. The use of plantain flour aims to diversify the consumption of the fruit, minimizing the large quantities currently lost during conveying, maturation, and commercialization. The use of plantain fruit in the unripe state is an interesting alternative, due to its high indigestible carbohydrate (components of dietary fiber) content, where resistant starch (RS) is the main component –4, the cExtrusion has been used to prepare pre-gelatinized starches that can be solubilized in water at room temperature. The increased solubility and overall digestibility of the pre-gelatinized starch are attributed to the disorganization of starch components in the granular structure, a process that can be enhanced as the gelatinization degree increases in the extruded starch. However, we hypothesized that if UPF is extruded under specific conditions, it can retain high RS content adding to non-starch polysaccharides in the fiber fraction, and may be thus included in foods where further cooking is not necessary, such as yogurt, smoothies, etc. Such a procedure would be in line with the current interest in the development of foods and ingredients with high dietary fiber contents, which derives from the evidences linking diverse health problems, e.g., overweight, obesity, diabetes, cardiovascular diseases, colon cancer, etc., to the generally low consumption of dietary fiber in occidental populations .The aim of this study was to produce a ready-to-use fiber-rich ingredient by extrusion of UPF, and to perform its chemical, functional and physicochemical characterization. The ingredient was also included in a yogurt to evaluate its impact on the rheological and sensory features of the final product.Unripe plantain fruits were harvested in Tuxtepe, Oaxaca, México. The flours from the pulp and from the pulp and peel were prepared as described by Ovando-Martinez et al.  and PatiUPF from pulp (UPFP) and the whole fruit (pulp + peel) (UPFW) were extruded in a single screw-extruder  at a constant rate of 75 rpm. The temperature in the three zones of the extruder  was kept constant at 50°C, conditions that in preliminary studies resulted in adequate rheological properties without promoting complete gelatinization of starch in plantain flours (unpublished data). Two batches were prepared for each type of flour.Thermal analysis of gelatinization (in excess water) was assessed to determine the temperatures and enthalpy associated to this phase transition. A 2.0 mg sample was mixed with 7 μl of deionized water. The wetted sample was equilibrated for 12 h at room temperature and subjected to the heating program over a temperature range from 20 to 100°C at a heating rate of 10°C/min in a DSC model 2010 . The temperatures of the phase transition and enthalpy (area under the phase transition curve) were calculated with the software of the equipment.The total dietary fiber (TDF) content of the raw and extruded samples was determined by the enzymatic method proposed by McCleary et al. , which yTotal starch (TS) was measured according to AACC method 76.13  using a _1. The temperature profile started with a heating ramp temperature of 5°C/min from 50 to 95°C, holding at 95°C for 10 min, cooling ramp temperature of 5°C/min from 95 to 60°C and finally holding at 60°C for 10 min. The starch concentration in the sample was 8% (dry basis).The pasting profile was evaluated during the cooking of the flours in a stress rheometer  using a starch pasting cell (SPC) with a vanned rotor at 500 sWater retention capacity was determined according to the method of Hallgren . BrieflyThe method described by Lin et al.  was usedWhere:−1 was chained.For viscosity measurements, an in-house made yogurt was mixed with plantain flours to yield total fiber concentrations ranging from 0.1 to 1.5% (w/v). Steady shear rate sweep was carried out at 25°C in a stress-controlled rheometer AR1500ex , using the standard concentric cylinders fixture . An ascendant sweep from 0.1 to 100 sin vitro digestibility was assessed according to the methodology described by Zheng et al. (2CO3) to prevent further amylase activity in the aliquot. After centrifugation , the glucose concentration in the supernatant was determined using a D-Glucose Assay Kit  according to the supplier instruction. Results are presented as starch hydrolyzed (grams per 100 g dry starch).The g et al. . In summOne hundred untrained judges evaluated the acceptability of the yogurt. Panelists were asked to assess their degree of liking using a 9-point hedonic scale, where 9 = like extremely and 1 = dislike extremely. The sensory evaluation was targeted to flavor, texture, odor and general acceptance of the yogurt, using the questionnaire proposed by Agama-Acevedo et al. .t-test was used for the statistical analysis of gelatinization degree.Results are presented as mean ± SD (standard deviation). Differences among the means obtained in each of the determinations were evaluated by one-way analysis of variance (ANOVA) with a significance level of α = 0.05, followed by Tukey's test using the statistical package Origin Pro 2016 . Student's The degree of gelatinization was determined in both extruded samples, the unripe plantain flour of the pulp (UPFP) and that from whole fruits (UPFW). The enthalpy and degree of gelatinization values are shown in Total starch (TS) content was higher in the flour of the pulp (UPFP) than in the peel-containing preparation (UPFW), and the same pattern was observed in the extruded samples . ExtrusiThe total dietary fiber (TDF) content of the flours, determined with a method for products “as eaten” , decreasThe high DF content recorded in both extruded samples, particularly UPFW, suggests the products' potential as functional ingredients that can be added to a variety of foods, like salads, fruits, smoothies, yogurt, and breakfast cereals. The possible use of the extrudates in other types of food such as salad dressings, emulsions, bakery products and snacks should be further explored.The pasting profile of both raw flours resembles those exhibited by pure starches , which aBoth raw flours showed similar water retention capacity (WRC) patterns, i.e., increased WRC concomitant with temperature increments . The higThe temperature-dependent solubility changes of all samples  followedThe raw flours showed similar OAI . The extR2 > 0.99 (k) was higher in the yogurt with extruded UPFW, which corroborated the larger viscosity increase this extruded preparation. It interesting to note that here-reported results on the effects of adding unripe plantain-derived flours to yogurts show an inverse pattern when compared to those reported for a yogurt added with resistant starch or β-glucans, where the n value decreased as the polysaccharide concentration increased  or the pulp (UPFP), produced ingredients rich in indigestible carbohydrates. The extruded UPFW exhibits higher fiber content (including resistant starch), water retention capacity and solubility than the extruded UPFP. The oil absorption index was relatively high in both extrudates. The addition of the extruded flours to a yogurt resulted in slightly increased viscosity, moderate starch hydrolysis rate and somewhat reduced acceptability of the product. Extruded UPFs represent a potential ingredient with nutritional-functional properties that may be used in ready-to-eat processed foods.LB-P and DG-V conceived the study. DG-V, LB-P, PF-S, and EA-A performed the experiments and participated in the acquisition of the data. DG-V, LB-P, PF-S, EA-A, and JT carried out the analysis and interpretation of the data. DG-V, LB-P, and JT drafted the manuscript. All authors read and approved the final version of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.