The adaptive evolution of timing of breeding (a component of phenology) in response to environmental change requires individual variation in phenotypic plasticity for selection to act upon. A major question is what processes generate this variation. Here we apply multi-year manipulations of perceived predation levels (PPL) in an avian predator-prey system, identifying phenotypic plasticity in phenology as a key component of alternative behavioral strategies with equal fitness payoffs. We show that under low-PPL, faster (versus slower) exploring birds breed late (versus early); the pattern is reversed under high-PPL, with breeding synchrony decreasing in conjunction. Timing of breeding affects reproductive success, yet behavioral types have equal fitness. The existence of alternative behavioral strategies thus explains variation in phenology and plasticity in reproductive behavior, which has implications for evolution in response to anthropogenic change. For phenotypic plasticity to evolve to a changing world, there must be variation in plasticity. Here, the authors show that whether great tits advance or delay breeding in response to perceived predation risk depends on their personality, linking variation in plasticity with that in personality. For example, anthropogenic change has made the climate more variable from year to year, thereby inducing selection for phenotypic plasticity in timing of breeding2. This adaptive evolution of population-level phenotypic plasticity requires individual variation in plasticity; therefore, predictions of population or species persistence will require insights in the ecological processes maintaining this type of variation.Phenotypic plasticity, the ability of organisms to adjust their phenotype to the environment, represents an important means by which organisms shift their phenology in response to environmental variation4 as it is postulated as a motivator for colonial and synchronous breeding8. However, a key unresolved question is whether synchronization is an ultimate adaptive strategy , or rather a proximal result of a common response to an environmental cue 9. Predators can induce seasonal increases in predation, and therefore may induce synchrony (or asynchrony) by nature of their hunting strategy. For example, the European sparrowhawk (Accipiter nisus) hunts by surprise attack and times its reproduction to coincide with the peak of fledging of their passerine prey, such as great tits (Parus major)3. Therefore, in the presence of sparrowhawk predators, great tit females may shift their timing of breeding in order to mismatch with the peak in sparrowhawk hunting. If all females respond by starting to breed at one particular time, synchrony may increase with predator presence. In our population, great tits respond to sparrowhawk cues  by decreasing time invested in conspicuous behaviors (singing), and increasing time invested in vigilance 10. Not all birds respond equally because individuals differ in heritable behaviors affecting predation risk, such as exploratory tendency assayed during short-term captivity . For example, in response to predator cues, individuals improve maneuverability by decreasing body mass11, which fast explorers  do more strongly than slow explorers12. Therefore, we predicted that all individuals do not respond the same to predator presence, thereby potentially decreasing breeding synchrony. We studied the consequences of these behavioral strategies for timing of breeding using replicated multi-year spatiotemporal manipulations in the wild.One of the key factors influencing optimal timing of breeding is predationTurdus merula), which is neither a predator nor a competitor of great tits. As a high-PPL treatment, we broadcast calls of the European sparrowhawk. Our playback frequency matched natural vocalization frequencies, and we broadcast on a 4 day on 4-day off scheme to avoid habituation10. Our playback design may have also altered individuals’ perception of temporal variance in risk, another factor influencing anti-predator behavior13, although competing theories debate whether mean rather than variance is important for prey to interpret predator cues14. Birds decreased risky communication behaviors (detailed above) in the high-PPL treatment but actual predator numbers were not affected (based on weekly counts)15, verifying that our manipulations influenced perceived—not actual—predation levels. In this vein, our aim with this study is to determine how behavioral types differ in their adjustment of reproductive investment in response to perceived levels.We manipulated perceived predation levels (PPLs) during spring and summer (March through July) among 12 nest box plots of great tits over a 2-year period Fig. . As a lo3, PPL manipulations should increase PPL particularly for peak and late-breeding great tits. Thus, only individuals that shift to breed earlier are likely to reap a pay-off in decreased risk. Moreover, owing to optimal shifts in how vigilance-foraging trade-offs are resolved, increased PPL should also generally decrease investment in foraging, increasing relative costs of egg production, and decrease clutch size4.Three plots received the low-PPL treatment in both years, three plots the high-PPL treatment in both years, and six plots changed treatment across years Fig. . This pa18. We thus propose here that individual plasticity in timing of breeding constitutes a key adaptive component facilitating the coexistence of these alternative behavioral strategies.As a null hypothesis, we considered that birds would not adjust timing of breeding as a function of PPL  was significantly below 1 (likelihood ratio test (LRT) for χ20/1 = 5.53, P = 0.01, Supplementary Table n = 172, LRT: χ20/1 = 10.52, P < 0.001; high PPL: n = 154, LRT: χ20/1 = 14.53, P < 0.001) but larger in the high-PPL treatment  was significantly reduced in the low PPL (n = 172) compared with the high PPL (n = 154) treatment  as a function of PPL treatment. The among-individual cross-context correlation between lay date expressed under low-PPL versus high-PPL  but did not differ between treatments , deviating from 0: LRT for χ21 = 14.74, P < 0.001, Supplementary Table χ20/1 = 1.97, P = 0.07, Supplementary Table F1, 22.4 = 0.01, P = 0.92, Supplementary Table Clutch sizes of our great tits responded differently than lay date to the treatments. Similar to lay date predictions, we predicted that individuals may alter their investment in current reproduction by changing clutch size in response to our manipulations. Either all individuals may respond the same to our manipulations, resulting in parallel reaction norms (as in scenario 1 but with negative slopes). Alternatively, predation danger may be highest only for the at-risk individuals, and therefore only these individuals would reduce clutch size. The among-individual variance in clutch size was significant in both treatments  compared with the high-PPL  treatment . In the high-PPL treatment, females were thus less able to reap the reproductive benefits associated with early breeding under lower PPL.In other great tit populations, females breeding early also produce larger clutchesion Fig. . The patn = 172) and the high PPL (n = 154) treatments, significant among-individual variance occurred in both treatments  and did not differ between treatments , deviating from 0  but not from 1 . However, among birds sharing the same lay date, faster explorers produced larger clutches . Importantly, the positive direct effect of exploratory behavior on clutch size canceled out the negative indirect effect on clutch size caused by faster explorers breeding late: the overall among-individual correlation between exploratory behavior and clutch size (Supplementary Table n = 172) and not significant .Path analyses revealed alternative behavioral strategies, related to exploratory tendency, individual plasticity in timing of breeding in response to PPL, and consequently, the existence of repeatable individual variation in timing of breeding within each PPL treatment Fig. . Under lβexploration → lay date ± SE: −0.15 ± 0.06, z = −2.45, P = 0.01, n = 154) . Among individuals sharing the same lay date, fast explorers also still produced larger clutches . Fast explorers nevertheless did not produce larger clutches overall: the among-individual correlation between exploratory behavior and clutch size .Under high PPL, fast explorers shifted forward relative to slow explorers and now initiated their clutches earlier than slow explorers 54) Fig. , which t Fig. , wβexploration → lay date, comparison of path coefficients between low PPL vs high PPL: t325 = 4.7, P < 0.001). Second, PPL significantly reduced the larger number of eggs that early breeders produced . Finally, PPL significantly reduced the larger number of eggs produced by fast explorers breeding at the same date as slow explorers . Consequently, the overall among-individual correlation between exploratory tendency and clutch size did not differ between treatments . Importantly, we used clutch size as a proxy for reproductive fitness as it has been shown to be a reliable indicator in other populations21. An alternative measure of fitness, the number of fledglings, as expected also did not vary as a function of exploratory tendency in either treatment group : low PPL: 0.04 , P = 0.43; high PPL: 0.07 , P = 0.23), implying that personality types, in fact, had equal reproductive success.Comparison of path coefficients revealed that PPL affected all paths by which exploratory behavior affected clutch size. First, PPL significantly advanced timing of breeding for faster relative to slower explorers  versus future reproduction 25. Fast-exploring great tits produced larger clutch sizes compared with slow-exploring great tits breeding at the same date  predation levels are now required to discover any fitness-related consequences of reproductive behavior mis-matching the environment28, as well as to fully address the fitness consequences associated with the alternative reproductive strategies revealed in this study. That is, while alternative personality types may indeed have equal reproductive success when predators are absent, the addition of seasonal or personality-related survival costs caused by predation may cause personality-related differences in fitness to arise when predators are present.Early breeding increased the number of eggs produced (clutch size), but neither clutch size nor reproductive success (fledging number) varied as function of exploratory tendency despite unambiguous links between timing of breeding and personality in both treatment groups Fig. . Treatme29, based on observational studies. Our study rejects this prediction experimentally, revealing that predator-induced asynchrony results from personality-related individualized responses to predation . Adult exploratory behavior was measured for each captured parent when nestlings were 7 or 9 days old, using a cage test adapted from a classic novel environment test39. See36 for a full description of the procedure. Briefly, each individual was recorded for 2 min; the sum of movements between different locations (scores ranged from 2 to 130) was used as a proxy of exploratory behavior. Values were scored later from the recording by an observer blind to the subject’s identity and treatment. This exploration score is a measure of activity that correlates with anti-predator boldness in our population thus we have validated its use as a proxy for risk-taking behavior in the face of predation threat. We performed 607 tests on 497 unique (ringed) birds. Of these, 387 were tested in only 1 year and 110 were tested in both years. Of the 110 birds with repeat measures, 29 individuals received the predator treatment both years, 32 received control both years, and 49 received both treatments.All work was ethically compliant with and carried out under Regierung von Oberbayern permit no. 55.2-1-54-2532-140-11. Data were collected in 2013 and 2014 in 12 forest plots that were established in a 10 × 15 km² area south-west of Munich, Germany37 Fig. . Each pl10 for full details). Four speakers  were evenly distributed across each plot in February and removed in July. For the first year of treatment (2013), assignment of treatments to plots was randomized, with the constraint that there be no initial differences between treatments in average breeding density, lay date, latitude, or longitude based on data from previous years. Six plots received a low-PPL treatment and six plots received a high-PPL treatment in the first year; the treatment was switched in half of the plots for the second year . In high-PPL plots, speakers were programmed with calls from sparrowhawks . Bird sounds were acquired from the Xeno-Canto database (www.xeno-canto.org) or provided by H. H. Bergmann. All speakers were programed to match the normal vocalization of our playback species: speakers broadcast approximately 60% of the time during the first 3 h after dawn and the last 3 h before dusk  and speakers broadcast approximately 15% of the time during the rest of the daylight hours (1.5 bouts per hour). The amount of silence between playback bouts was determined randomly to avoid habituation. Playback was broadcast at 90 dB . Sparrowhawks are resident predators—they stay in the area of their nest and hunt over a wide territory surrounding it40. This means that presence of a sparrowhawk during the breeding season (like our sound cues) should signal potential predation risk throughout the rest of the season to prey. In addition, a single observation of a predator is known to have a long lasting effect on prey, as it is more difficult to assess absence of a predator, whereas the costs of mis-assessment are higher14. Therefore, playback was given for 4 consecutive days (on), followed by 4 consecutive days of non-playback (speakers were off), the cycle was repeated throughout the season; this design prevented habituation10 without decreasing the effectiveness of the high-PPL manipulation. Data were not biased by dispersal events as only 0.13% of birds  have moved between plots in our years of collecting data (2010–2017), and no birds moved between treatment plots during the years of our study.We conducted a playback experiment in order to manipulate PPLs  as treatment varied at this level, thereby avoiding pseudo-replicated values of P for effects of treatment18. Random intercepts were also fitted for individual identity, where female  identity was assigned in analyses of lay date and clutch size because our previous work showed that female rather than male identity determines such life-history traits18. Exploratory behavior, by contrast, was measured for each individual parent separately, and individual identity effects thus estimated using data from both sexes. In some populations, exploratory behavior varies between sexes and over the course of the day the analysis of this behavior therefore also included sex  and time of day  as two additional fixed effects. These univariate analyses thus partitioned the total phenotypic variance , clutch size, or exploratory behavior, as the focal response variable (Supplementary Table R) were calculated for each random effect as the variance attributable to the focal effect divided by the total phenotypic variance not attributable to fixed effects , the value of Ps was calculated assuming an equal mixture of χ2(0) and χ2(1) because variances are bound to be zero or positive48 .The significance of fixed effects was based on the We used bivariate mixed-effects models to estimate the pattern of among-individual variation in plasticity in response to PPL treatment for each of the three phenotypic traits  separately  and covariances (Cov):These bivariate mixed-effects assumed a bivariate normal distribution estimating all level-specific variances  calculated as x represents the focal hierarchical level of interest. The phenotypic correlation in the data between measurements of a focal trait in the low-PPL versus the high-PPL treatment  and χ2(1) because correlations deviating from the value 1 can do so only by being lower (not higher) than 148 (χ20/1 in Supplementary Table The statistical significance of the among-individual cross-context correlation was assessed using a LRT, calculated as twice the difference in log likelihood between the full model and a model where mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimP associated with each path was calculated using z-tests. We used t-tests to compare estimates from the two treatment groups.We used a tri-variate version of the mixed-effects model detailed above to estimate among-individual correlations (entclass1pt{minimaFurther information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


There is a paucity of information about how human hippocampal subfields are functionally connected to each other and to neighbouring extra-hippocampal cortices. In particular, little is known about whether patterns of functional connectivity (FC) differ down the anterior-posterior axis of each subfield. Here, using high resolution structural MRI we delineated the hippocampal subfields in healthy young adults. This included the CA fields, separating DG/CA4 from CA3, separating the pre/parasubiculum from the subiculum, and also segmenting the uncus. We then used high resolution resting state functional MRI to interrogate FC. We first analysed the FC of each hippocampal subfield in its entirety, in terms of FC with other subfields and with the neighbouring regions, namely entorhinal, perirhinal, posterior parahippocampal and retrosplenial cortices. Next, we analysed FC for different portions of each hippocampal subfield along its anterior-posterior axis, in terms of FC between different parts of a subfield, FC with other subfield portions, and FC of each subfield portion with the neighbouring cortical regions of interest. We found that intrinsic functional connectivity between the subfields aligned generally with the tri-synaptic circuit but also extended beyond it. Our findings also revealed that patterns of functional connectivity between the subfields and neighbouring cortical areas differed markedly along the anterior-posterior axis of each hippocampal subfield. Overall, these results contribute to ongoing efforts to characterise human hippocampal subfield connectivity, with implications for understanding hippocampal function. •High resolution resting state functional MRI scans were collected.•We investigated functional connectivity (FC) of human hippocampal subfields.•We specifically examined FC along the anterior-posterior axis of subfields.•FC between subfields extended beyond the canonical tri-synaptic circuit.•Different portions of subfields showed different patterns of FC with neocortex. AAnterior (of the hippocampus)ABAnterior body (of the hippocampus)CA 1-4Cornu ammonis 1-4DGDentate gyrusDMNDefault mode networkENTEntorhinal cortexFCFunctional connectivityPBPosterior body (of the hippocampus)PHCPosterior parahippocampal cortexPRCPerirhinal cortexRSCRetrosplenial cortexrsfMRIResting state functional magnetic resonance imagingTTail (of the hippocampus)1The hippocampus has been implicated in supporting multiple cognitive functions including episodic memory , the imaThe primary input to the hippocampus is via the entorhinal cortex (ENT), the source of the canonical tri-synaptic pathway. The ENT primarily innervates the DG and, from here, intra-hippocampal connectivity is generally acknowledged to follow a unidirectional pathway through the CA regions to the subiculum, the primary region of efferent projection from the hippocampus . While tOne way to investigate homologues of these anatomical frameworks in vivo in the human brain is to characterise patterns of functional connectivity (FC) using resting state functional magnetic resonance imaging (rsfMRI). While rsfMRI FC often reflects anatomical pathways, its statistical dependencies are not limited to the underlying anatomy , 2010. TOf the extant rsfMRI studies that considered human hippocampal subfield FC in some shape or form, several focussed on intra-medial temporal lobe (MTL) FC, including hippocampal subfields. They observed that activity was highly correlated between hippocampal subfields, and that functional networks within the MTL may divide into separate ‘functional modules’, one broadly consisting of hippocampal subfields and another consisting of parahippocampal regions . Other sThere have been some reports that patterns of FC for each hippocampal subfield broadly reflect those observed in studies of whole hippocampus network connectivity . By contOf the very small number of studies examining FC in different parts of a subfield, it has been reported that the anterior portions of human subiculum and CA1 showed preferential FC with PRC while posterior portions exhibited preferential FC with PHC . Vos de In the current study we aimed to systematically characterise not only the FC of each hippocampal subfield in its entirety  but also the FC of different portions of each subfield along the anterior-posterior axis of the human hippocampus. We used a combination of high resolution structural MR imaging and high resolution rsfMRI to examine FC between the subfields themselves – DG/CA4, CA3/2, CA1, subiculum, pre- and parasubiculum, and the uncus – and subfield FC with key neighbouring regions in the memory/navigation network, specifically, ENT, PRC, PHC and also the RSC, due to its well documented anatomical connectivity with specific hippocampal subregions, in particular, the subiculum, pre- and parasubiculum .Of note, previous studies of hippocampal subfield FC have generally incorporated the entire subicular complex into a single subiculum mask . All gave written informed consent to participate in accordance with the University College London research ethics committee.2.2Structural and functional MRI data were acquired using a 3T Siemens Trio scanner  with a 32-channel head coil within a partial volume centred on the temporal lobe that included the entire extent of the temporal lobes and our other ROIs.Structural images were collected using a single-slab 3D T2-weighted turbo spin echo sequence with variable flip angles (SPACE)  in combiFunctional data were acquired using a 3D echo planar imaging (EPI) sequence which has been demonstrated to yield improved BOLD sensitivity compared to 2D EPI acquisitions . Image rwww.fil.ion.ac.uk/spm). All images were first bias-corrected, to compensate for image inhomogeneity associated with the 32 channel head coil , into 3 (for DG/CA4 and CA3/2) or into 2 (for the uncus) separate sections along its longitudinal axis (anterior (A), anterior body (AB), posterior body (PB) and tail (T); see 2.4http://atlas.brain-map.org to gain insights into the likely location of the RSC. The RSC mask reflected our best attempt to remain as faithful as possible to the histological investigations of RSC. It was restricted to the thin strip of cortical tissue lying on the ventral bank of the cingulate gyrus and posterior to the corpus callosum, as described in detail by The ENT, PRC and PHC were segmented using the guidelines laid out by 2.5http://www.nitrc.org/projects/conn. The data were temporally bandpass filtered (0.01–0.1 Hz) and corrected for white matter and ventricular signal. To create FC matrices, time series of voxels within each of the ROIs were averaged and correlated with the averaged time series of all other ROIs resulting in correlation coefficients which were then transformed using Fisher's z calculation. Rather than using simple bivariate correlations, we used semi-partial correlations which allowed us to identify the ‘unique’ contribution of a given source on a target area. Of note, semi-partial correlations are computed between unmodified and other residualised variables, essentially regressing out or controlling contributions of other additional variables. Therefore, for each seed analysis in turn, slightly different values were regressed out, resulting in test statistics that vary marginally in their magnitude. That is, the semi-partial correlations between source region A and target region B might be slightly different than the semi-partial correlation between source region B and target region A. The resulting semi-partial ROI-to-ROI correlation matrices from the native space first-level analyses were further averaged at the second level in order to examine group effects. Importantly, this ROI-to-ROI approach allowed us to test hypotheses regarding FC between each ROI and all other ROIs using minimally preprocessed data . This approach minimised the mixing of BOLD signal between adjacent subfields. Any functional voxels overlapping an ROI border were assigned to whichever ROI contained the majority of its volume. Inevitably, there were differences between subfields, and portions of subfields, in terms of the number of functional voxels, given their size differences. What relationship this has, if any, to the number of functional connections identified or the strength of those connections is currently unknown. This issue should be explored in future studies.We used the CONN toolbox version 14 for rsfMRI data analysis First, we conducted ‘whole subfield’ analyses with 10 bilateral ROIs .We then conducted ‘longitudinal axis’ analyses with 24 bilateral ROIs . For both sets of analyses, ROI-to-ROI results were corrected for multiple comparisons and reported when significant at a level of p < 0.05 false discovery rate (FDR) corrected .33.1We first analysed the FC of each hippocampal subfield in its entirety, in terms of FC with other subfields and with the cortical ROIs. The results are summarised in DG/CA4 was significantly correlated with CA3/2, CA1, subiculum, uncus, PHC and RSC.CA3/2 was correlated with DG/CA4 and the pre/parasubiculum.CA1 was correlated with DG/CA4, subiculum, uncus, CA3/2 and PHC.Subiculum was correlated with pre/parasubiculum, CA1, DG/CA4, ENT and PRC.Pre/parasubiculum was correlated with the subiculum, CA3/2, uncus, ENT, PHC and RSC.uncus was correlated with CA1, pre/parasubiculum and PHC.The These results suggest that each hippocampal subfield had a unique pattern of FC with other hippocampal subfields, and that each subfield showed a different pattern of FC with the cortical ROIs.3.2Next, we analysed FC for different portions of each hippocampal subfield along its anterior-posterior axis, in terms of FC between different portions of a subfield, FC with other subfield portions and with the cortical ROIs. These results are summarised in 3.2.1Activity in the AB portion was significantly correlated with activity in A CA1, AB CA3/2, AB CA1, AB subiculum, AB uncus, PB DG/CA4, T subiculum, PHC and RSC.The PB portion was associated with AB DG/CA4, PB CA3/2, PB CA1, PB subiculum, T DG/CA4.The T portion was associated with PB DG/CA4, T CA3/2, T CA1 and T subiculum.To summarise, in relation to FC between different portions of DG/CA4, each part was correlated with adjacent but not distant portions (e.g. the AB portion correlated with the PB portion but not the T portion). Considering FC with other subfields, the DG/CA4 in each portion of the hippocampus correlated with CA3/2, CA1 and subiculum within the same portion of the hippocampus, but rarely with more distant portions of these subfields. AB DG/CA4 was the only portion of the DG/CA4 to correlate with more distant portions of other subfields, specifically the A CA1 and T subiculum. AB DG/CA4 also correlated with AB uncus. In addition, AB DG/CA4 was the only portion of the DG/CA4 to correlate with the cortical ROIs, specifically PHC and RSC. This suggests that AB DG/CA4 may have a broader pattern of both intra- and extra-hippocampal FC compared with more posterior portions.3.2.2Activity in the AB portion was correlated with A CA1, AB DG/CA4, PB CA3/2 and PB pre/parasubiculum.The PB portion was associated with AB CA3/2, PB DG/CA4 and T CA3/2.The T portion was correlated with PB CA3/2, PB pre/parasubiculum, T DG/CA4 and T CA1.To summarise, in relation to FC between different portions of CA3/2, each part was correlated with adjacent but not distant portions. Considering FC with other subfields, CA3/2 in each portion of the hippocampus correlated with DG/CA4 within the same portion of the hippocampus. The AB CA3/2 correlated with A CA1. The T CA3/2 correlated with the T CA1 and both the AB and T CA3/2 were correlated with the PB pre/parasubiculum. We found no evidence for CA3/2 correlations with any of the cortical ROIs.3.2.3The A portion was associated with A uncus, AB DG/CA4, AB CA3/2, AB uncus and T CA1.The AB portion was associated with AB DG/CA4, AB subiculum, PB CA1.The PB portion was associated with AB CA1, AB subiculum, PB DG/CA4, PB CA3/2, PB subiculum, T CA1 and PHC.The T portion was associated with A CA1, PB CA1, T DG/CA4, T CA3/2,T subiculum and RSC.To summarise, in relation to FC between different portions of CA1, AB and PB portions of CA1 were correlated only with adjacent portions. A and T CA1 were correlated with each other. Considering FC with other subfields, CA1 in each portion of the hippocampus correlated with DG/CA4 and subiculum within the same portion of the hippocampus but rarely with distant portions of these subfields. The only parts of CA1 to show FC with the cortical ROIs were PB CA1 with PHC and T CA1 with RSC.3.2.4The A portion was associated with AB subiculum, A pre/parasubiculum, A uncus and ENT.The AB portion was correlated with A CA1, A subiculum, AB DG/CA4, AB CA1, AB pre/parasubiculum, AB uncus, PB CA1, PB subiculum, ENT and PRC.The PB portion was associated with AB subiculum, PB DG/CA4, PB CA1, PB pre/parasubiculum, T subiculum and T pre/parasubiculum.The T portion was associated with AB DG/CA4, PB subiculum, T DG/CA4, T CA1 and T pre/parasubiculum.To summarise, in relation to FC between different portions of the subiculum, each portion was correlated with adjacent but not distant portions. Considering FC with other subfields, the subiculum in each portion of the hippocampus correlated with DG/CA4, CA1 and pre/parasubiculum within the same portion of the hippocampus, but also often with distant portions of these subfields. In terms of FC with the cortical ROIs, A and AB subiculum correlated with ENT while AB subiculum correlated with PRC.3.2.5The A portion was associated with A subiculum, A uncus and AB pre/parasubiculum.The AB portion was correlated with A pre/parasubiculum, A uncus, AB subiculum, AB uncus, PB pre/parasubiculum, PHC and RSC.The PB portion was associated with AB CA3/2, AB pre/parasubiculum, PB subiculum, T CA3/2, T pre/parasubiculum, PHC and RSC.The T portion was associated with PB subiculum, PB pre/parasubiculum, T subiculum and RSC.To summarise, in relation to FC between different portions of the pre/parasubiculum, each part was correlated with adjacent but not distant portions. Considering FC with other subfields, the pre/parasubiculum in each portion of the hippocampus correlated with subiculum only within the same portion of the hippocampus, with the exception of the T pre/parasubiculum which also correlated with PB subiculum. PB pre/parasubiculum correlated with AB and T portions of the CA3/2. The A pre/parasubiuculum correlated with A uncus while AB pre/parasubiculum correlated with both A and AB uncus. In terms of FC with the cortical ROIs, PB pre/parasubiculum correlated with PHC while AB, PB and T pre/parasubiculum correlated with RSC.3.2.6The A portion was associated with A CA1, A subiculum, A pre/parasubiculum, AB uncus and PRC.The AB portion was associated with A CA1, A uncus, AB DG/CA4, AB subiculum, AB pre/parasubiculum and PHC.To summarise, in relation to FC between different portions of the uncus, the A and AB portions were correlated with each other. Considering FC with other subfields, both the A and AB uncus correlated with subiculum and pre/parasubiculum within the same portion of the hippocampus. Both A and AB uncus correlated with A CA1 while AB uncus correlated with AB DG/CA4. In terms of FC with the cortical ROIs, A uncus correlated with PRC while AB uncus correlated with PHC.4In this study we investigated the resting state FC of hippocampal subfields and specific extra-hippocampal ROIs by leveraging high resolution structural and functional MRI. We first probed the FC of each hippocampal subfield in its entirety . We examined how each subfield interacted with other hippocampal subfields and with neighbouring cortical areas. We then investigated the FC of the anterior, anterior body, posterior body and tail portions of each subfield. We aimed to characterise how each portion of each subfield interacted with other portions of the same subfield, with other portions of other subfields and, finally, with neighbouring cortical ROIs. Our results provide new insights into how human hippocampal subfields may functionally interact with each other along the anterior-posterior axis of the hippocampus during a resting state. In addition, we show that the ENT, PRC, PHC and RSC preferentially interact, not only with specific hippocampal subfields, but with specific portions of each subfield along the hippocampal longitudinal axis.4.1Previous studies investigating FC between hippocampal subfields have found them to be highly correlated with each other . In the For the most part, we observed functional homologues for the well characterised canonical intra-hippocampal anatomical circuitry (DG/CA4 → CA3/2 → CA1 → subiculum) in alignment with our predictions . However, in addition, we also documented some unexpected functional correlations. For instance, we observed an association between CA3/2 and pre/parasubiculum. A rationale for a functional association between these regions is unclear, and we are unaware of any previous reports of direct anatomical or functional interactions between the pre/parasubiculum and CA3/2 in the human brain. These observations suggest that FC between hippocampal subfields may extend beyond the current understanding of anatomical connectivity. We return to this point later.4.2In addition to having different patterns of FC with other subfields, each hippocampal subfield had a unique pattern of FC with the neighbouring ROIs. Largely aligning with our predictions, the ENT was associated with the subiculum and pre/parasubiculum; the PRC was associated with the subiculum; the PHC with the DG/CA4, CA1, pre/parasubiculum and uncus; and the RSC with the pre/parasubiculum and DG/CA4.This is the first study to investigate FC of the pre/parasubiculum in the human brain. Concordant with one of our primary predictions, activity in the pre/parasubiculum was correlated with activity in RSC. The RSC is a key node of the parieto-medial temporal visuospatial processing pathway  and sendWe have previously outlined a potential rationale for the functional significance of the pre/parasubiculum in visuospatial processing in relation to scene-based cognition . In briePrevious studies of hippocampal FC do not differentiate between the pre/parasubiculum and subiculum ‘proper’  and, theThe interpretation of other interesting observations from the whole subfield analyses are facilitated by the results of the longitudinal axis analyses. We therefore discuss other elements of these results in the context of the longitudinal axis analyses below.4.3Considering the longitudinal axis analyses, we first investigated how each portion of each subfield interacted with other portions of the same subfield . We observed that, within each subfield, not all portions were functionally correlated with each other, as would be expected if each subfield was functionally homogeneous. Rather, generally within each subfield, adjacent portions were correlated while distant portions were not. For example, AB DG/CA4 correlated with the adjacent PB DG/CA4 but not with the more distant T DG/CA4. This pattern was consistent for almost all subfields and implies a functional heterogeneity within each hippocampal subfield and that distant portions of each subfield may not be functionally ‘in synch’ with each other. These results mirror patterns of intra-subfield anatomical connectivity recently described by 4.4We next investigated how each portion of each subfield interacted with different portions of other subfields. In accordance with our predictions, the functional homologues for the canonical anatomical framework observed in the whole subfield analysis were, for the most part, also observed within each portion of the hippocampus (e.g. DG/CA4 was correlated with CA3/2 within AB, PB and T portions of the hippocampus; see Taken together, these observations suggest that while, for the most part, different portions of each hippocampal subfield have FC with associated subfields in the same portion of the hippocampus, specific portions of each subfield may have preferential FC with distant rather than nearby portions of associated subfields. From an anatomical perspective, such interactions may be facilitated by longitudinal fibres which project through the anterior-posterior axis of the hippocampus, supporting functional links between distant portions of hippocampal subfields .4.5The longitudinal axis analyses offered additional insights into the nature of the FC patterns we identified at the whole subfield level in terms of FC with the neighbouring cortical ROIs. We found evidence that different portions of each subfield had different patterns of FC with these cortical regions. We discuss each of these observations in turn.The results of our longitudinal axis analyses broadly support previous FC studies which suggested that the PRC and PHC disproportionately interact with anterior and posterior portions of the hippocampus respectively , particuIt is important to note that while we specifically predicted that PRC would be correlated with anterior portions of CA1 and subiculum, neuroanatomical evidence suggests a link between the PRC and the transition zone of the CA1 and subiculum, encompassing a region sometimes referred to as the prosubiculum. This CA1-subiculum transition area has been consistently observed in numerous tract tracing studies in the non-human primate and appears to have direct interactions with a number of brain regions including the PRC and cingulate cortex , suggestAs noted above, previous FC studies also suggest that PHC has preferential connectivity with posterior portions of CA1 and subiculum. Concordant with this, we observed a correlation between PB CA1 and PHC. In contrast, however, we did not observe an association between the subiculum and PHC. Rather, we noted that the AB and PB portions of the pre/parasubiculum were correlated with PHC. A reason for this discrepancy may lie in how the subiculum has been defined in previous studies where, as alluded to earlier, pre/parasubiculum is typically incorporated into a general subiculum region. By separating these areas, our data provide increased specificity on the nature of this functional interaction.An interesting pattern was also observed in the A and AB portions of the uncus. The A uncus was associated with PRC while the more posteriorly located AB uncus was associated with PHC. To our knowledge, there are no previous studies of the FC of the human uncus and, therefore, this is the first report of a functional dissociation between its anterior and posterior portions. It must be noted that the uncus is a complex portion of the hippocampus which itself contains multiple subfields  that canAnterior portions of the subiculum were correlated with ENT, reflecting the well characterised anatomical connections between these regions . It was Finally, we observed correlations between the RSC and the AB, PB and T portions of the pre/parasubiculum and the T portion of the CA1. Anatomical frameworks of the parieto-medial temporal visuospatial processing pathway, described earlier, highlight that the RSC sends direct projections that specifically innervate the pre/parasubiculum and CA1 portions of the hippocampus . Our FC It is important to note that the results of these longitudinal analyses show that correlations found at the whole subfield level may be driven by specific portions of a subfield rather than the whole. For example, the whole subfield analyses revealed that the pre/parasubiculum was correlated with CA3/2 while the longitudinal analysis revealed, more specifically, that the PB portion of the pre/parasubiculum was correlated with AB and T portions of CA3/2 while no other portion of the preparasubiculum was correlated with CA3/2. In a similar manner, while the whole subfield analyses revealed that the CA1 was correlated with PHC, the longitudinal analyses suggest that only the PB portion of CA1 was correlated with PHC. Patterns such as these underline the overarching theme of our results, namely that different portions of each subfield have different patterns of FC both with other hippocampal subfields and with neighbouring cortical ROI's and that, where possible, this should be taken into account in future studies.4.6We do not claim that our results represent the only patterns of communication between these anatomically complex regions. Moreover, while we investigated FC of broad portions of each subfield, we do not suggest that FC is segregated in such a coarse manner. Rather, the gradient nature of connectivity along hippocampal subfields is well documented in both the anatomical  and funcapriori ROIs. Also related to methodology, the placement of hippocampal subfield boundaries on MRI is an ongoing area of research yet to gain a consensus. Different hippocampal segmentation schemes have different criteria for subfield boundary placement (Our results complement previous studies which have observed functional gradients along the long axis of hippocampal subfields . It is ilacement  and alsolacement . The resIn conclusion, we have provided evidence that different hippocampal subfields have different patterns of FC with each other and with neighbouring cortical brain regions and, importantly, that this is also the case for different portions of each subfield. These differential patterns of FC may be facilitated, as we propose is the case for the association between the pre/parasubiculum and RSC, by anatomical connections which bypass the canonical hippocampal circuitry, permitting direct interactions to occur. This suggests that anterior and posterior portions of hippocampal subfields may be incorporated into different cortical networks which, in turn, might provide potential mechanisms by which functional differentiation along the long axis of the hippocampus can occur. Further investigations are required to assess the validity of this interpretation, and to investigate how these patterns of FC may be modulated by individual differences, normal ageing, in the context of brain pathologies and during the performance of different cognitive tasks.


International Journal of Oral Science (2015) 7, 205–212; 10.1038/ijos.2015.29; published 18 September 2015Correction to: In the original version of this Article, Fig.


Nature Communications; 10.1038/s41467-018-05461-5, published online 06 August 2018Correction to: a (3a0), plate thickness (0.4a0), and radius of perforated holes r (0.18a0) but different hole-center distance characterized by b.’ The correct version states ‘plate thickness (a0)’ in place of ‘plate thickness (0.4a0)’ and ‘radius of perforated holes r (a0)’ rather than ‘radius of perforated holes r (0.18a0)’.The original version of this Article contained errors in the second sentence in the legend of Fig. 1, which incorrectly read ‘These two elastic insulators are identical in lattice constant −3) with a fixed plate thickness 7.82 mm.’ In the corrected version, ‘mass density 7903 kg m−3’ replaces ‘mass density 7803 kg m−3’.The first sentence of the ‘Sample preparation’ section of the Methods originally incorrectly read ‘Our samples are prepared exclusively on polished stainless-steel plates , lattice constant a  and radius of perforated holes r  maintain constant.’ The correct version states ‘√3 × 0.4a0’ in place of ‘0.4a0’ and ‘√3 × 0.18a0’ rather than ‘0.18a0’.The second sentence in the legend of Supplementary Fig. 3, originally incorrectly read ‘The symmetry of the phononic crystal remains unchanged as CThis has been corrected in both the PDF and HTML versions of the Article.


Structural Dynamics, was integral and valued in a way that words cannot express. She was the key person in the foundation of the relationship between AIP Publishing and the American Crystallographic Association, one of AIP's member societies. A long-time member of the ACA and the crystallography community, Judith spent 35 years as a small molecule crystallographer at the Naval Research Laboratory in Washington, D.C., and later worked for Rutgers University in New Jersey on a project for the Protein Data Bank. She was co-editor of the magazine ACA RefleXions and a former editor of the International Union of Crystallography (IUCr) Newsletter. She was a past chair of the U.S. National Committee for Crystallography, served as ACA president in 1991, and was named to its inaugural class of Fellows in 2011.It is with a heavy heart that we learned of the passing of Judith on March 31, 2018. Her role with our journal, Structural Dynamics—an open access title launched jointly with AIP Publishing in 2014. It was during this time where I got to know Judith, as she was the person who interviewed me for the position of Editor-in-Chief on behalf of the ACA. From this first meeting, it was immediately clear that there was accord in our vision for the journal. We understood each other very well, and I was very pleased by how much our views echoed each other. Judith was very supportive of my vision for the journal and accepted the suggestion to name the journal Structural Dynamics. Most of all, I remember her constant support and encouragement of strategic initiatives. I was extremely pleased to be accompanied by such a constructive partner in the challenging but fascinating adventure of launching a new journal.Judith served as ACA's representative on the AIP Publishing Board of Managers since its inception in 2013 and was AIP's corporate secretary. In this respect, she was the driving force behind the creation of ACA's first journal, Structural Dynamics were displayed for ACA members. Judith was wonderful in the way she took the destiny of the journal to heart. I recall how excited and happy she was when the journal achieved its first Impact Factor of 3.667 in 2015 and how thrilled she would have been to know the latest Impact Factor of 3.969. The journal and our team owe Judith a debt of gratitude. May she rest in peace.The evolution of the journal was closely followed by Judith, as she was an active member of the team participating in conference calls, attending conferences, and commissioning prominent authors to publish in the journal. In addition, Judith's commitment to promoting the journal was repeatedly showcased in ACA's quarterly newsletter, ACA RefleXions, where featured articles of


This study evaluated the combined effect of recipient-to-donor weight and sex mismatch after deceased-donor renal transplantation in a German transplant cohort and the evolution of recipient-to-donor weight difference over a 13-year observation period. The association of absolute weight and sex difference with graft failure was explored in an outpatient cohort of deceased-donor transplant recipients who underwent kidney transplantation between 2000 and 2012. Graft failure was defined as repeated need for dialysis or death with a functioning graft. Recipient and donor sex pairings were classified as sex concordant (MDMR/FDFR) or discordant (MDFR/FDMR). These classes were further stratified into four groups according to recipient-to-donor weight mismatch ≥10 kg (recipient > donor) or <10 kg (recipient < donor). Multivariable Cox proportional hazards models were applied to evaluate the time to graft loss adjusting for donor, immunologic, surgical, organizational, and recipient predictors. Sex-concordant transplant pairings <10 kg weight difference served as the reference group. Among 826 transplant recipients, 154 developed graft failure (18.6%). Median graft survival time was 3.9 years; first quartile (0.2–1.2), second quartile (1.2–2.9), third quartile (2.9–5.8), and fourth quartile (5.8–12.4). After multivariable adjustment, the highest relative hazard for graft failure was observed for sex-discordant transplant pairings with a ≥10 kg weight difference between recipient and donor . A recipient-to-donor weight mismatch of ≥10 kg was associated with an increased risk of graft loss or recipient death with a functioning graft. Concurrent sex discordance seemed to enhance this effect as indicated by an increase in the hazard ratio. We detected no significant tendency for increasing recipient-to-donor weight differences from 2000 to 2012. Renal transplantation has become the primary option for the treatment of end-stage renal disease in many countries. Over the past decades, kidney transplant recipients of organs from deceased donors have benefitted from improvements in survival rates and quality of life compared with end-stage renal disease patients on dialysis . NeverthThis study was approved by the local ethics committee (Ethikkommission der Medizinischen Fakultät der Heinrich Heine Universität Düsseldorf number 6200R). Data were coded in a manner that ensured subjects could not be identified either directly or through linked identifiers. Since this study involved retrospective review of existing data, Institutional Review Board  approval was obtained, but without specific informed consent from the patients. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The need for informed consent was waived due to the retrospective nature of the research.This analysis represents a cohort study of all patients receiving a deceased-donor solitary kidney transplant in our clinic between January 1, 2000 and December 31, 2012. Recipients of organs from living donors, patients younger than 18 years of age, those receiving multiple organs, and those with missing or implausible data were excluded.We defined the primary exposure as a combination of donor-recipient weight and sex mismatch. Sex pairing between donor and recipient was categorized as either sex-identical female donor to female recipient and male donor to male recipient (FDFR & MDMR) or sex-disparate (FDMR & MDFR). Absolute weight difference between the recipient (R) and the donor (D) was categorized as <10 kg and ≥10 kg (R to D). These absolute weight difference thresholds were recently reported as clinically relevant in a large sentinel investigation of the SRTR . Each reThe outcome of interest was graft failure or loss for any reason. Graft failure was defined as the need for chronic dialysis or repeat pre-emptive transplantation or death with a functioning graft.eurotransplant.orgwww.).In addition to the primary exposure, previously reported and presumed predictors of graft loss including donor and recipient height, donor and recipient weight, donor and recipient age, donor and recipient BMI, cold ischemia time, warm ischemia time, dialysis vintage, categories of human leukocyte antigen mismatch (0–6), panel reactive antibody category , cytomegalovirus risk constellation, recipient hepatitis C and B virus status, recipient diabetes mellitus status, and whether transplants were performed under the Eurotransplant Senior Programme were analyzed. All data were extracted from clinical charts or electronic databases, including the hospital's laboratory database and the Eurotransplant's electronic resource  were graphically displayed for each donor-recipient sex/weight pairing compared with the reference group. We further performed unadjusted and adjusted multivariable Cox proportional hazards models to test for an association of donor-to-recipient weight and donor-to-recipient sex difference with graft loss . Finally, we searched for an effect of BSA on the primary outcome. Here, two categories of BSA were analyzed in two different approaches, < vs. ≥0.01 mFig 1). Among these recipients, 23.5% were ≥10 kg larger than the donors, and 49.5% of the transplants were performed using a sex-discordant graft. Mean recipient weight was 68.7 ± 11.9 kg, and mean donor weight was 82.9 ± 11.4. Mean and median absolute weight difference between the recipient and the donor were -5.4 kg, and -6.0 kg, respectively. Females accounted for 40.3% of the recipients and 48.4% of the donors. Additional baseline characteristics stratified according to sex and weight pairing are shown in The initial cohort consisted of 982 deceased-donor renal transplant recipients. We excluded 130 donors due to missing graft outcome data, and 26 were excluded due to incomplete datasets. Therefore, we analyzed 826 complete datasets . Although there was a tendency for enlarged interquartile ranges and increasing numbers of outliers, we did not observe a statistically significant increase in weight difference.Of the 826 individuals included in this analysis, graft failure occurred in 154 patients (18.6%). Mean follow-up time was 3.8 ± 3.0 years and median follow-up time was 2.9 years . The risk of graft failure for each donor-recipient sex pairing in our cohort was higher when the weight of the recipient was greater than that of the donor. In multivariable Cox regression analysis, the risk of graft loss was highest among recipients of sex-disparate transplants who had a concurrent weight mismatch of ≥10 kg R to D , relative to sex-identical transplants with no weight mismatch  without yielding significant results on uni-  as well as multivariable Cox-regression analyses .In the unadjusted analysis the risk of graft loss in settings where the recipient's weight was ≥10 kg higher than that of the donor was 1.63 , whereas the adjusted analysis showed an even greater risk . In contrast to weight mismatch, sex mismatch was not predictive of graft failure in the unadjusted analysis  or the adjusted test . Further details are shown in 2 and < vs. ≥0.20 m2 BSA difference from recipient to donor, neither of which yielded significant results on multivariable analysis (data not shown).As BSA has also been reported to predict graft loss, we performed an identical statistical analysis using an exposure variable of combined recipient-to-donor sex and BSA mismatch. Here, two categories of BSA were analyzed with two different approaches; < vs. ≥0.01 mA significant impact of recipient factors on donor graft outcome has been observed in the past . RecentlSeveral studies reported negative graft outcomes in clinical situations involving small kidney donors in relation to the recipient ,7,14,15.Sex mismatch has also been reported to potentially contribute to increased sensitization after transplantation, with subsequently reduced graft outcome. It has been postulated that mismatch between the H and Y minor histocompatibility antigens  promotes allograft rejection or de novo donor-specific antibody evolution with subsequent deterioration of transplant function and graft loss ,24,25. IAlthough current techniques in routine clinical practice still do not allow for meaningful nephron counting, promising new techniques are in development ,30,31. HFurther, we could not identify a massive increase in recipient and donor weight mismatch from 2000 to 2013, although the prevalence of a BMI over 30 in three large German cohorts  rose sigThere are several limitations to this study. The most important difference to the sentinel investigation by Miller and colleagues  lies in We confirmed the negative impact of recipient-to-donor weight mismatch on graft survival in a German cohort of deceased-donor transplant recipients, and this effect seemed to be enhanced by sex-discordant transplantation. Weight and sex mismatch and their combined effect should be considered in future investigations of long-term graft outcome to elucidate any possible positive and/or negative effects that may be relevant to the implementation of graft allocation systems. Here, expected positive effects on graft longevity must be weighed carefully against possible negative effects on wait times for organ donation in particular patient subgroups.S1 File(SAV)Click here for additional data file.


Spain has dramatically increased the number of controlled circulatory death donors (cDCD). The initial selection criteria for considering cDCD for kidney transplantation (KT) have been expanded progressively, with practically no limits in donor age during the last years. We aimed to analyze the early clinical outcomes using expanded (> 65 years) cDCD in comparison with standard ones.Observational multicenter study including 19 transplant centers in Spain. We performed a systematic inclusion in a central database of every KT from expanded cDCD at each participant unit from January-2012 to January-2017. Surgical procedures and immunosuppressive protocols were based on local practices. Data was analyzed in the central office using logistic and Cox regression or competitive-risk models for multivariate analysis. Median time of follow-up was 18.1 months.p < 0.001) and with higher comorbidity. At 1 year, no differences were found amongst older and younger cDCD KT recipients in terms of serum creatinine (1.6 (SD 0.7) vs 1.5 (SD 0.8) mg/dl; p = 0.29). Non-death censored graft survival was inferior, but death-censored graft survival was not different . They also presented a trend towards higher delayed graft function  but a similar rate of primary non-function , and acute rejection . In the multivariate analysis, in short follow-up, donor age was not related with worse survival or poor kidney function (eGFR < 30 ml/min).561 KT were performed with kidneys from cDCD, 135 from donors older than 65 years. As expected, recipients from older cDCD were also older (65.8 (SD 8.8) vs 53.7 (SD 11.4) years; The use of kidneys from expanded cDCD is increasing for older and comorbid patients. Short-term graft outcomes are similar for expanded and standard cDCD, so they constitute a good-enough source of kidneys to improve the options of KT wait-listed patients. Kidney transplantation (KT) from donors after cardiac death (DCD) might imply poorer graft outcomes, as circulatory death constitutes an injury to the organs that may result in lower graft survival . To miniIn Spain, improvements in organ procurement and assessment tools have allowed us to expand donor acceptance criteria . ParticuIn 2012, Spain developed a strategy to encourage the use of controlled DCD (cDCD) for transplantation, a modality less used in our environment because of the high rate of brain-death donation . That reAs donor age in this modality has also experienced a substantial change during the last years, we aimed to analyze the early results obtained with KT from elderly cDCD donors, both in terms of early clinical outcomes – primary non function (PNF) and delayed graft function (DGF) – as well as 1st year patient and graft survival, in order to reassure with the strategy of using these kidneys for aged recipients or change the policy if needed.Observational and multi-center registry, including 19 transplant centers in Spain. Data from all cDCD (Maastricht type III) KT performed from January 2012 to January 2017 were collected and prospectively recorded by nephrologists at each center database following the same structure. For this analysis, a data manager central office merged anonymous databases in a single one. Informed consent for kidney transplantation and local electronic data management was obtained according to each center’s Institutional Review Board policy, and a central research Ethics Committee of H.U. Puerta de Hierro approved the project. A pre-defined analysis after the first 500 cDCD KT was performed in order to evaluate the early results obtained with kidneys from donors over 65 years, and to establish strategy changes if needed. This age cut-point was chosen based on the upper limit recommended by the 1st version of Spanish National Transplant Organization cDCD strategy guides in 2012 ), while dialysis vintage longer than 24 months prior to KT conditioned DGF . However, death-censored graft survival was similar in both groups   showed a lower 1st-year-graft survival among those kidneys from cDCD donors> 65 years . Nine patients from the group of older donors died with functioning graft during the first year after transplantation (mostly in the first 6 months after transplantation) due to cardiovascular events (n = 4), sudden death of uncertain origin (n = 1), cancer (n = 1), sepsis (n = 1), pulmonary embolism (n = 1), and unknown cause (n = 1). Again, the multivariate analysis revealed that only recipient age was as risk factor for patient survival, increasing the risk around 10% per each recipient’s year of age, both with Cox and competing risk analyses , we found that older donors with one or two of those previous risk factors associated with significant higher DGF rate  allocated to a high-risk recipient cohort. We found a higher mortality compared to recipients who received organs from younger donors. However, in the multivariate model the recipient age accounted for all the risk and the weight of the donor age over the death risk disappeared. Three different multivariate statistical models showed these results, which reflects their consistency. On the other hand, short-term graft survival was similar and donor age itself did not have any impact in patient or graft outcomes.Our results pointed at similar patient and KT short-term outcomes regardless of donor age, which may suggest better results than those previously reported. If these findings were confirmed in long-term studies, they might generate changes in acceptance and allocation of these grafts for KT. In fact, when we added comorbidity factors other than donors’ age, some outcomes as DGF seem to be affected. Careful pre-transplant evaluation should be performed in these extremely high-risk group in order to improve outcomes, but kidneys from old cDCD should not be discarded systematically.


Tumor-to-tumor metastasis (TTM) is a rare but well-documented phenomenon that is defined as metastasis in a histologically distinct tumor. Ovarian mature teratomas (OMTs) can coexist with various cancers by malignant transformation, which may make it difficult to distinguish these from TTM. Herein, we report a case of TTM from appendiceal adenocarcinoma to the OMT, mimicking the malignant transformation of OMT.A 67-year-old Japanese woman underwent abdominal total hysterectomy and bilateral salpingo-oophorectomy for an ovarian tumor in another hospital. She was initially diagnosed with mucinous carcinoma/carcinoid arising in the OMT. One year after surgery, she was referred to our hospital after the presentation of increased appendiceal mass. Cecal biopsy targeting an appendiceal tumor revealed scattered mucinous cells with signet ring features, which were morphologically similar to the malignant components in the previously diagnosed right OMT. Both the appendiceal adenocarcinoma and malignant components of the OMT stained positive for CK7, CK20, CDX-2, and SATB2 but negative for estrogen receptor, progesterone receptor, and pax-8. Finally, we confirmed the diagnosis of appendiceal goblet cell carcinoid metastasizing to the right OMT. The patient had tumor-bearing survival due to systemic chemotherapy administered for 35 months after the initial surgery.Awareness of the TTM phenomenon is important to avoid an incorrect diagnosis and to select the appropriate therapy when unusual malignancy is encountered in the OMTs. Ovarian mature teratomas (OMTs) are common tumors that account for 20% of all ovarian neoplasms. An OMT is a germ cell tumor composed of cells derived from at least two, but frequently all three, germ cell layers. Although OMTs are usually benign, 1–3% undergo malignant transformation [Tumor-to-tumor metastasis (TTM) is a rare but well-documented entity that is defined as a metastasis in a histologically distinct tumor. This phenomenon was first reported in 1902, and since then, approximately 200 cases have been reported . The mosA 67-year-old Japanese woman  with no past cancer history had visited another hospital with lower right abdominal pain. Enhanced computed tomography revealed the presence of a 70-mm mass with non-uniform enhancement at the right ovary and a 10-mm enlarged appendix with strong enhancement, for both of which OMT and appendicitis were suggested , and the absence of gastrointestinal epithelium in OMT. Mucinous carcinoid of the ovary histologically resembles mucinous/goblet cell carcinoid of the appendix. Baker et al.  reportedINK4a and in situ hybridization of human papillomavirus supports the diagnosis of a cervical cancer origin [TTM is a well-documented phenomenon, for which a misdiagnosis of the primary organ may occur. TTM involving an ovarian tumor as the recipient is unusual, with only 13 cases described to date (Table r origin .Table 1In conclusion, we report a case of TTM wherein an appendiceal adenocarcinoma metastasized to an OMT, mimicking the malignant transformation of OMT. To the best of our knowledge, the present case is the first case of TTM due to the metastasis of appendiceal adenocarcinoma to an OMT. Awareness of this phenomenon is important to avoid an incorrect diagnosis and to select the appropriate therapy when unusual malignancies in OMTs are encountered.


Solanum tuberosum L.) are a good dietary source of carbohydrates in the form of digestible starch (DS) and resistant starch (RS). As increased RS content consumption can be associated with decreased chronic disease risk, breeding efforts have focused on identifying potato varieties with higher RS content, which requires high-throughput analysis of starch profiles. For this purpose, freeze drying of potatoes has been used but this approach leads to inaccurate RS values. The present study objective was to assess the starch content ) of three cooked potato genotypes that were dried using freeze drying and innovative drying techniques  relative to freshly cooked potato samples. Depending on the genotype, all drying methods showed one or more starch measures that were significantly different from freshly cooked values. The combination of ultrasound and infrared assisted conductive hydro-drying was the only method identified to be associated with accurate assessment of DS and TS content relative to fresh samples. The drying treatments were all generally associated with highly variable RS content relative to fresh controls. We conclude that freshly cooked samples must be used for selecting varieties with a high proportion of RS starch as drying of cooked potatoes leads to unreliable RS measurements.Potatoes ( Solanum tuberosum L.) are an important worldwide staple food crop, which serves as a good dietary source of carbohydrates, vitamin C, several B vitamins, antioxidants, minerals and protein [Potatoes  and resistant starch (RS) content . The DS Freeze-drying (FD) has commonly been used prior to starch quality assessment for both research and industry . LyophilAlternative drying technologies to FD are required for high throughput sample processing of cooked potatoes for accurate determination of starch quality. In that regard, recent novel food drying technologies have been developed as an alternative to FD, that are equivalent or superior in terms of preservation of heat-sensitive nutritional components. These include instant controlled pressure drop drying , microwave vacuum drying (MVD) and conductive hydro-drying (CHD). The DDS process involves subjecting a sample to multiple rounds of pressure-drops until a desired moisture content is achieved . Swell dThe present study involved an investigation of the capacity of the above innovative drying processes to contribute to accurate assessment of starch digestibility of dried cooked potatoes in comparison to digestibility measures from freshly cooked potato samples. The tested drying methods included FD, DDS, MVD and CHD. Three well-characterized potato genotypes were subjected to in vitro starch digestion to assess for the DS, RS and TS content of the cooked potato samples after undergoing various drying treatments. Fluorescence microscopy and scanning electron microscopy (SM) were carried out to visually assess the effects of the drying methods on starch granule integrity.Organically grown potato tubers from three genotypes were obtained from the Bon Accord Seed farm operated by Potatoes NB . The genotypes ‘Russet Burbank’ (RB), ‘Atlantic’ (ALT) and ‘Yukon Gold’ (YG) were used. Thirty pounds (13.6 kg) of each genotype were obtained and subsequently stored in a cold room (4–10 °C) until use.Potato genotypes were individually processed and analyzed. For each genotype, the potatoes were washed and whole tubers were separated into four replicates, with ten tubers per replicate. For each replicate, potato tubers were cooked in boiling water until they reached acceptable softness, defined as when a stainless-steel knife could easily penetrate the tubers, which was validated with a meat thermometer indicating an internal temperature above 90 °C . Upon coBefore and after each drying treatment, the cooked tubers were weighed, which was calculated as previously described . The calFor each genotype and replicate, the cooked potato material was equally divided into five main treatments: fresh (control), FD, MVD, DDS and CHD. CHD was further subdivided into four treatments: CHD using a 82 °C water bath and infrared light (CHD1), CHD at 82 °C using an ultrasound water bath (CHD2), a combination of the two above CHD treatments , also known as ultrasound and infrared assisted conductive hydro-drying (UIACHD). Additionally, standard conductive hydro-drying using 95 °C water bath (CHD4) was tested. Between 10–20 g of cooked potato material was dried per treatment. A small fraction (~1–5 g) of each replicate and treatment was saved for microscopy. Material that was not used for immediate starch analysis was stored at −80 °C until analysis. For each drying treatment, samples were dried completely.® tubing fittings. The DDS drying consisted of 720 cycles of pressure variation per hour over 6 h, for a total of 4320 cycles. During each cycle, a primary (outer) valve was opened for 1 s to allow the pressure to increase within the chamber to 75 psi. Afterwards, the value was closed for 1 s and the samples slightly heated. The secondary (inner) valve was opened for 1 s, causing a pressure drop within the chamber and then closed for 2 s. The total drying cycle lasted 5 s and was completed at 27.5 °C. Once MVD and DDS drying was completed, the materials were sent back to McGill University and these were ground to a powder with a coffee grinder and stored at −80 °C until starch analysis.For the fresh treatment, starch analysis on cooked tubers was completed immediately after 24 h cooling to ensure complete retrogradation of starch. FD samples were completed at −50 to −60 °C and 0.85 mBar (0.64 mm Hg)  using previously established conditions . After dFour different CHD setups of a batch laboratory scale ultrasound and/or infrared assisted conductive hydro-dryer were constructed according to References ,38. Setuw/w RS.Starch content  was assessed using the Megazyme Resistant Starch assay kit (K-RSTAR)  as described previously . The assAspergillus niger amyloglucosidase; AMG) for 16 h at 37 °C in a shaking water bath  at 200 strokes/min. Samples were then centrifuged at 1500× g for 10 min and the supernatant (DS portion) and pellet (RS portion) separated. DS samples were diluted to 100 mL with 100 mM sodium acetate buffer (pH 4.5). Aliquots of 0.1 mL with 10 µL of AMG (300 U/mL) were incubated for 20 min at 50 °C. Afterwards, 3.0 mL reagent enzyme mixture , was added and further incubated for 20 min at 50 °C. D-Glucose content was determined by measuring the absorbance with a spectrophotometer  at 510 nm. The buffer and GOPOD reagent were used as blank and D-glucose (1 mg/mL), as standards for starch content determination. The pellet (RS portion) was washed once with 99% v/v ethanol and twice with 50% v/v ethanol. For each wash, the samples were centrifuged at 1500× g for 10 min and the supernatant decanted. The pellet (RS portion) was resuspended using 2 M KOH buffer for 20 min in an ice water bath, on a magnetic stirrer. Exactly 0.1 mL of AMG (3300 U/mL) was added along with 8 mL of sodium acetate buffer (1.2 M) and 100 μL of AMG (3300 U/mL) and incubated for 30 min in a 50 °C water bath. After centrifugation, aliquots of 0.1 mL were treated with 3.0 mL of GOPOD reagent and incubated for 20 min at 50 °C. D-Glucose content was measured as described above for the DS portion. Each absorbance measurement was completed in duplicate. The glucose content of the collected supernatant and the digested pellet was calculated as per the kit instructions and summed (DS + RS) to calculate total starch (TS) content. Calculations included the conversion of absorbance to glucose content, weight and volume correction and a factor to convert the measured D-glucose content to anhydro-D-glucose that occurs in starch. For each genotype and treatments, 4 biological replicates were used (n = 4). All starch content was calculated on a dry mass basis in terms of a 100 g portion size (g/100 g DW), then calculated as % difference from the fresh control.In brief, samples (100 mg of the dried sample or 0.5 g of the fresh sample) were digested with 4.0 mL of pancreatic α-amylase containing dilute Fluorescent microscopy was used to visualize the surface structure of freshly cooked and cooled potato as well as the dried samples. In brief, each sample was transferred to glass slides with 2 drops of distilled water. To look at starch granule surfaces, a single drop of Calcofluor-white (1 g/L)  was added to the slide and allowed to react for 2 min. To view granule surfaces, the slides were viewed under dark field with an excitation wavelength of 365 nm. Image collection was performed at magnifications of 175× with a photomicroscope assembly  imaging software).A Hitachi TM3000  SM was used to visualize the surface structure of dried potato samples. A small layer of dried potato powder was mounted on a thin layer of carbon tape. Images were captured using TM3000 software  using “Compo” and “Shadow1” image modes at 100×, 500× and 1000× magnification and obtained at 5 kV.p < 0.05. Data was reported as the mean % difference between treatment and control ± standard error of the mean (SEM) and p < 0.05 was considered significant.Statistical analyses were completed using Jmp Pro 13.2.1  and figures using Origin(Pro) . Within each genotype, Dunnett’s test was used to compare drying treatments with the fresh treatment as the control. Outliers were determined using Grubbs’ test and excluded from the dataset if p < 0.05) in comparison to fresh samples (p < 0.05) relative to controls by 26.5 ± 4.6, 25.9 ± 1.4% and 27.0 ± 2.6% for genotypes ALT, RB and YG respectively. DDS treatment was not significantly different from the fresh control in the genotype YG but DS content was significantly (p < 0.05) lower by 23.5 ± 7.0% and 34.5 ± 1.1% in ALT and RB, respectively. No significant changes in DS content were observed for all genotypes after CHD, except for CHD2 which led to an underestimation of DS (p < 0.05) in RB by 10.2 ± 1.5%. For all DS measurements, the observed differences were greater than the sensitivity of the kit used  was shown have underestimated RS content in YG by 42.9 ± 6.1%, whereas RS content was underestimated for every genotype with the CHD2 and CHD4 treatments. CHD3 showed no statistical difference between the fresh controls for ALT, RB and YG. For all RS measurements, the observed differences were greater than the sensitivity of the kit used  after FD . No signused see . The staThe calculated percent differences in RS content between the fresh controls and drying treatments appeared to be the most variable, as compared to DS and TS . For exap < 0.05) overestimated by 12.3 ± 4.0% (p < 0.05) underestimation of TS content. Specially, the TS values after MVD were significantly underestimated (p < 0.05) for ALT, RB and YG relative to controls by 17.0 ± 8.3%, 25.3 ± 1.9%, 24.9 ± 3.3%, respectively. DDS treatment led to underestimated TS content in ALT and RB by 19.5 ± 7.1% and 31.1 ± 1.1%, respectively whereas no statistical difference was found in YG.As seen with DS measurements, the effect of drying on TS content varied by genotype. No statistical difference between the fresh control and FD were observed with ALT and YG, although the TS content of RB was significantly  measurements were affected by drying treatments in a genotypic-dependent manner. This finding is mostly likely due to inherent differences in starch structure and content that can vary between genotypes ,25,26, wAll the CHD treatment combinations showed genotype-dependent differences in RS, DS and TS. CHD3, otherwise known as UIACHD, showed no statistical differences in DS, RS and TS compared to control. Although not significant, the percent difference in RS using CHD3 was still high 34.9 ± 7.7%). Future studies, however, could consider the use of UIACHD towards drying of cooked potatoes for high-throughput screening of DS or TS content as these measures showed minimal variations relative to the fresh controls. Microscopic analysis showed that the structural integrity of potato samples after UIACHD was significantly less disturbed than FD, although both methods showed differences in comparison to fresh samples. UIACHD can be considered as a possible alternative to FD as UIACHD is faster, more energy efficient and less costly ,34. Due .9 ± 7.7%After investigation of multiple non-conventional drying methods, it is apparent that for accuracy of starch profile measurements, use of freshly cooked samples is still important, particularly with respect to RS content. The UIACHD method, however, shows promise as a drying process towards accurate evaluation of the DS and TS content of cooked potatoes and so this technology could be further investigated in that regard. Screening genotypes for optimal starch profiles using fresh cooked potato samples is a difficult process due to the seasonal demands of harvested food crops, particularly since storage time/conditions are confounding variables that affect the nutritional content of potatoes. Hence, identification of other drying alternatives for high-throughput RS analysis is a key next step to support the commendable initiatives by potato breeders to identify table stock with improved starch profiles for consumers.


Grandparents raising grandchildren experience multiple challenges as they take on the unexpected role of caring for their grandchildren, which usually occurs under stressful and stigmatizing conditions. Many of the challenges grandparents experience are well documented in the research. Less attention is given to understanding how a grandparent caregiver’s social network changes when s/he becomes a caregiver and how her/his social network influences resilience. Thus, the purpose of this study was to use social network analysis (SNA) to examine the relation between social networks and resilience in grandparents raising their grandchildren. This was done by conducting face-to-face interviews with twenty grandparents raising grandchildren after they completed a survey measuring social support, social isolation, and resilience. The interview protocol included questions related to participants’ social network, social support, and services. Prior to the interviews, using data from the surveys participants were identified as representing one of four resilience quadrants: resilient, maladaptive, competent, and vulnerable. Qualitative analysis of grandparent’s social networks across groups indicated resilient grandparent caregivers’ networks were structured in a way that provided more opportunities for the inflow of new information and resources. Whereas the proportion of professionals in maladaptive grandparent caregivers’ networks tended to be less than for other networks. This could suggest that for grandparent caregivers, having professionals in one’s network can be beneficial. Findings from the current study provide opportunities for future research such as identifying ways to help grandparent caregivers structure their social networks to promote resilience.


Many cannabinoid actions in the nervous system are mediated by CBin vivo and mouse embryonic stem cell differentiation in vitro.We investigated the pro-neurogenic potential of the synthetic cannabigerol derivative, VCE-003.2, in striatal neurodegeneration by using adeno-associated viral expression of mutant huntingtin Oral administration of VCE-003.2 protected striatal medium spiny neurons from mutant huntingtin-induced damage, attenuated neuroinflammation and improved motor performance. VCE-003.2 bioavailability was characterized and the potential undesired side effects were evaluated by analyzing hepatotoxicity after chronic treatment. VCE-003.2 promoted subventricular zone progenitor mobilization, increased doublecortin-positive migrating neuroblasts towards the injured area, and enhanced effective neurogenesis. Moreover, we demonstrated the proneurogenic activity of VCE-003.2 in embryonic stem cells. VCE-003.2 was able to increase neuroblast formation and striatal-like CTIP2-mediated neurogenesis.The cannabigerol derivative VCE-003.2 improves subventricular zone-derived neurogenesis in response to mutant huntingtin-induced neurodegeneration, and is neuroprotective by oral administration.The online version of this article (10.1186/s40035-019-0148-x) contains supplementary material, which is available to authorized users. Huntington’s disease (HD) is a monogenic neurodegenerative disease produced by the expression of mutant huntingtin (htt) protein with expanded glutamine repeats in the N-terminal portion of the protein . Mutant Cannabis sativa, and their endogenous counterparts 2-arachidonoylglycerol and anandamide exert symptomatic relief in various neurodegenerative and neuroinflammatory conditions . 3-NP-treated animals and their respective non-lesioned controls (injected with PBS) were used for pharmacological studies with VCE-003.2. Treatments consisted of oral gavage every 24 h with VCE-003.2 at a dose of 10 mg/kg using sesame oil as vehicle. 12 h after the last injection of 3-NP motor activity, hindlimb clasping and kyphosis were evaluated as previously described [


As usual to each New Year, the annual review of Iran J Public Health, hereby is presented for the year 2018. Besides, this editorial will compare the trend of whole publication process during 2015–2018.The total number of manuscripts received during 2018 was 2540 from 60 countries. Of course, only the country of corresponding author was considered, so altogether much more countries we had in the panel. Again, Iran had the highest rate of submission, followed by China and South Korea  Fig. 1)Fig. 1). Out of total submission of 2540 articles during 2018, 2005 articles were rejected after initial in-house evaluation or later peer review. Therefore the acceptance rate for this year was 15.2%.In 2018, still we had unfortunately noticed some cases of plagiarism, which were treated according to the journal policy and COPE instructions. Normally, authors of minor cases of plagiarism are given a chance to amend their manuscripts precisely but major cases are rejected.The Journal continued its policy as for exact peer review rules including in-house evaluation followed by double blind peer review system. The reasons for rejecting a manuscript during in-house evaluation are various but the most important cases are out of scope cases, poor outcome, local studies, clinical contents etc. The types of articles published during 2015–2018 are shown in Due to high flow of submitted manuscripts, in many cases, the authors were requested to change the format of “Original Article” to “Letter to the Editor”, which of course the merit of both formats remains the same.http://www.scimagojr.com/, the H index of the journal is 24 until 2017. Besides, SCOPUS has reported the Site Score of the journal as 0.85 for 2017, besides SNIP as 0.6788.According to The good news for us and respected authors was that the Impact Factor announced by Clarivate Analytics (ISI) for the year 2017 was 1.053. In comparison to previous years, fortunately we had a great improvement on the journal quality. We should respect all referees and authors from all over the world to cooperate with us sincerely and caused their journal to further its aims. We do hope in near future, to improve this rate as much as possible . This fi


Nursing & Health Sciences Research Journal (NHSRJ). This article discusses how it was developed, the role of the librarians, and future goals of the journal.Librarians at Baptist Health South Florida (BHSF) launched the first peer-reviewed journal from BHSF,  Nursing & Health Sciences Research Journal (NHSRJ). NHSRJ provides an open access platform for health care providers to disseminate research findings and improve patient care  is a not-for-profit health care organization that operates numerous hospitals, outpatient centers, and outreach programs in south Florida. The Library and Knowledge Services Department includes five site libraries and provides library services and electronic resources to employees and affiliated physicians system-wide. In March 2018, we launched the first peer-reviewed journal from BHSF, ent care .Digital Commons from bepress, includes a publishing module. It includes out-of-the box tools to manage the entire article submission, peer-review, and acceptance process, along with the tools needed to host an online journal. The platform is cloud-based; is highly customizable in look, feel, and organization; and does not require coding skills beyond basic hypertext markup language (HTML).In 2015, we began discussions with stakeholders at BHSF about the possibility of launching a peer-reviewed journal. The technical capability to provide publishing support services was already in place because the platform for our existing institutional repository, Since the platform was already budgeted for and licensed by our department and we had recently added staffing to support the project with the hire of a full-time medical librarian, we felt confident that we had the resources necessary to offer this exciting new service line. There is a high level of interest in research dissemination at BHSF, given that several of our hospitals have Magnet status and the high volume of research projects conducted by our health care professionals and staff system-wide. Therefore, we were not surprised by the enthusiastic response that we got from various stakeholders.The first to partner with the library was the nurse scientist team from the Nursing and Health Sciences Research Department. The library serves as publisher in this partnership. We provide the platform to host and archive the e-journal and all associated support services including copyright advice, training, search engine optimization, journal and site layout, metadata creation, loading of content, and application for an International Standard Serial Number (ISSN). Future plans include obtaining digital object identifies (DOI) assignments to identify journal content and submitting applications for indexing in applicable databases. The nurse scientist team and editorial board is responsible for editorial and submission policies, engagement in marketing communications, and all content, including solicitation of submissions, management of the peer-review process, and copyediting.While increasingly common in university library settings, providing publishing services remains rare in hospital libraries that are not university affiliated. We enjoyed the opportunity to join our university counterparts and learn about the technical aspects of publishing a journal. We also gained a deeper knowledge of editorial best practices and learned through benchmarking and experience about the importance of clearly defining the scope of services and in drafting service-level agreements for our stakeholders. The result of offering this new service line is that we have raised our visibility and found a new way to demonstrate our value to our organization. The feedback has been overwhelmingly positive, and we look forward to continuing to support publications at BHSF.


NegFluo was applied to Arabidopsis leaves of wild-type and ss4 mutant plants. We validated its accuracy by comparing morphological quantifications using NegFluo and state-of-the-art methods relying either on starch granule purification or on preparation-intensive electron microscopy combined with manual image analysis. NegFluo thus opens the way to fast in situ analysis of starch granules.Starch granules that accumulate in the plastids of plants vary in size, shape, phosphate, or protein content according to their botanical origin. Depending on their size, the applications in food and nonfood industries differ. Being able to master starch granule size for a specific plant, without alteration of other characteristics , is challenging. The development of a simple and effective screening method to determine the size and shape of starch granules in a plant population is therefore of prime interest. In this study, we propose a new method, NegFluo, that combines negative confocal autofluorescence imaging in leaf and machine learning (ML)-based image analysis. It provides a fast, automated, and easy-to-use pipeline for both  Understanding the initial steps of starch synthesis would open the route to control the number of starch granules produced in each plastid and consequently their size for a given carbon flux: the fewer initiation events, the larger the granules.Starch is a polysaccharide that accumulates in plants and is synthesized in either photosynthetic or storage organs. In leaves, starch accumulates during the day and is degraded at night to provide carbon and energy for the plant in the absence of photosynthetic activity . The polArabidopsis thaliana  already described in −2 s−1. Seeds were incubated at 4°C in 0.1% Phytagel solution (w/v) for 3 days before being sown on peat-based compost.sis.info ]. ColumbSample preparation was adapted from the protocol described in ex = 488 nm and λem = 500–550 nm (green channel) and with λex = 561 nm and λem = 570–620 nm (red channel). To further increase the contrast between leaf autofluorescence and starch darkness, median filtering and contrast-limited adaptative histogram equalization  was applied using an ImageJ macro before the ML step  with a Plan Apo 60x Oil (NA = 1.4) objective. The autofluorescence was acquired with λ ML step .The ML approach is based on the “Waikato Environment for Knowledge Analysis” (WEKA) implemented in ImageJ . We firsin situ visualization of segmented starch granules. These three additional pipeline steps ensure that starch granules that are out of focus or at the edge of the image are excluded from statistical analysis. From extracted parameters, mean values and standard errors were calculated and plotted using Excel (Microsoft). Standard error does not represent a measurement error but corresponds to the diversity of starch granules size.Automated analysis allows screening through a high number of images. Thus, we chose to analyze starch granules with the highest confidence level possible. Probability maps were first thresholded to an 80% confidence interval in each pixel for both starch granules and plastids. To reinforce the accuracy, we developed an ImageJ macro combining (i) various binary operations to ensure that detected starch granules were completely embedded into plastids, (ii) watershed operations to separate adjacent granules , and  equipped with a 20-mm aperture tube are fully described in4), and embedded in Epon resin. The blocks were cut with a diamond knife in a Leica UC6 ultramicrotome. Backscattered electron images of the non-conductive surface were recorded under a low pressure of air (100 Pa) in a FEI Quanta 250 FEG environmental scanning electron microscope (ESEM) operating at 7 kV.Leaf fragments were fixed with glutaraldehyde, post-fixed with osmium tetroxide (OsO4 and embedded in Epon resin. About 70-nm-thin sections were cut with a diamond knife in a Leica UC6 ultramicrotome and post-stained with periodic acid–thiosemicarbazide–silver proteinate (PATAg) (Samples were prepared as described in  (PATAg) . SamplesIn situ starch granule imaging performed on fresh leaves is quite challenging. Indeed, most plastid components generate a high autofluorescence with a global broad excitation and emission spectrum. Thus, traditional strategies involving tagging, like iodine staining  and, for practical reasons, fixed with paraformaldehyde  and the aldehyde Figure 1pii1 mutant as described by These samples were then observed through NegFluo and compared with traditional electron microscopy imaging. As shown in To characterize the starch granules in more details, one needs to measure their size and shape. However, manual segmentation is time-consuming and not adapted to analyze a large number of starch granules. While NegFluo provides ease to visually analyze images, extracting robust parameters to automatically discriminate between the different structures of the leaf is not trivial using parametric approaches. We thus developed an ML-based pipeline to circumvent this technical limitation.We developed the procedure based on Trainable Weka Segmentation module, since it combines the benefits of (i) FiJi  for imagMoreover, its implementation is modular and transparent, which allowed us to control the different steps as well as the possibility of customizing the algorithms and their evaluation . The Neg(i) The initial image : edge deArabidopsis leaf images were manually labeled in several classes. We tested configuration ranging from two classes (starch granules and non-starch granules) to five classes . The optimal configuration consisted of defining four classes: the starch granule, the plastid, the cytosol delimited by cell membrane, and the background. Below four classes, the algorithm could not properly discriminate between starch granules and background, while five classes increased the number of pixels inaccurately attributed between these two dark-pixel structures. Different classifiers were also available. We selected the fast random forest . It required a relatively low number of images for training (20 labeled images), was polyvalent, and avoided overfitting, and its computational cost for training was low. Part of the image pixels was then used for training, while the remaining pixels were subject to the algorithm to validate its accuracy ((ii) Dozens of (iii) Once the classifier was trained, it was applied to new images and provided four probability maps. Thus, each pixel was assigned with a probability to be part of each category. Pixel color corresponds to the higher probability between the starch granule (red), the plastid (green), the membranes (blue), and the background (white) ((iv) From the probability maps, one could apply a traditional segmentation algorithm. To ensure optimal quality of starch detection, we developed an ImageJ macro (a) segmenting pixels with a probability higher than 80%, (b) segmenting starch granule with starch pixels embedded in plastids pixels, and (c) separating adjacent starch granules based on the watershed algorithm. Morphological properties of starch granules were then extracted from the segmented images , while the average starch granule areas are significantly different between Col-0 and ss4 using both methods (p < 0.001 with both techniques). Starch granules size was also determined using a Coulter counter, and the results were compared with those obtained by NegFluo-ML. Again, the results are consistent between the two methods, and the differences between the diameters of the Col-0 and ss4 starch granules can be highlighted by both techniquesTo ensure the relevance of these measurements, it was required to compare morphological parameters extracted from NegFluo-ML to state-of-the-art starch analysis methods. We thus analyzed images from Col-0 and Measurements performed using NegFluo-ML were in perfect agreement with those obtained with previously used methods without the need to embed the samples in a resin for electron microscopy or to extract starch granules for Coulter analysis.in situ starch granule morphological characterization. Previous works using confocal imaging for in situ starch granule observation  and the University of Lille. We thank Agence Nationale de la Recherche for funding .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.