Stress treatment was started when all plants exhibited detectable tubers. Combined heat and drought stress was applied by increasing growth temperature for 2 weeks and simultaneously decreasing daily water supply. CT analysis revealed that tuber growth is inhibited under stress within a week and can resume after the stress has been terminated. After cessation of stress, tubers started growing again and were only slightly and insignificantly smaller than control tubers at the end of the experimental period. These growth characteristics were accompanied by corresponding changes in gene expression and activity of enzymes relevant for starch metabolism which is the driving force for tuber growth. Gene expression and activity of Sucrose Synthase (SuSy) reaffirmed the detrimental impact of the stress on starch biosynthesis. Perception of the stress treatment by the tubers was confirmed by gene expression analysis of potential stress marker genes whose applicability for potato tubers is further discussed. We established a semi-automatic imaging pipeline to analyze potato tuber delevopment in a medium thoughput (5 min per pot). The imaging pipeline presented here can be scaled up to be used in high-throughput phenotyping systems. However, the combination with automated data processing is the key to generate objective data accelerating breeding efforts to improve abiotic stress tolerance of potato genotypes.As a consequence of climate change, heat waves in combination with extended drought periods will be an increasing threat to crop yield. Therefore, breeding stress tolerant crop plants is an urgent need. Breeding for stress tolerance has benefited from large scale phenotyping, enabling non-invasive, continuous monitoring of plant growth. In case of potato, this is compromised by the fact that tubers grow belowground, making phenotyping of tuber development a challenging task. To determine the growth dynamics of tubers before, during and after stress treatment is nearly impossible with traditional destructive harvesting approaches. In contrast, X-ray Computed Tomography (CT) offers the opportunity to access belowground growth processes. In this study, potato tuber development from initiation until harvest was monitored by CT analysis for five different genotypes under stress conditions. Tuber growth was monitored three times per week  Solanum tuberosum L.) is the fourth most important crop plant worldwide and as such of great significance with respect to food security  . Potato Conventional methods to follow tuber growth over time involved the removal of tubers and/or alterations in the substrate leading to potential disturbances of source-sink relationships . X-ray cin vivo and analyzed gene expression of potato tubers with destructive sampling.Here we describe the implementation of a medium-throughput imaging pipeline enabling us to continuously monitor growth of potato tubers by means of CT analysis. We observed the implications of combined heat and drought stress on tuber growth velocity Solanum tuberosum plantlets of the cultivars Agria, Saturna, Tomensa, and Ramses were obtained from Solana Research GmbH . The cultivar Diamant was obtained from KWS Potato BV . All plantlets were propagated in tissue culture on MS-Medium (–2 s–1) and 8 h dark at 21°C. Plants were transferred to individual pots with 15 cm diameter and a volume of 1.5 L containing sieved soil . The pots were placed in plant growth chambers  under conditions of 16 h light at 21°C and 8 h dark at 18°C and 50% humidity during the day and 35% humidity at night. Plants were watered daily with 50 ml per day/plant and tuber growth was monitored by CT three times per week. In the first experiment, four plants each of the cultivars Agria, Saturna, Tomensa, Ramses and Diamant were monitored at the same time. When tubers were detectable via CT, combined heat and drought stress was applied by increasing the temperature to 29°C during the light period and 21°C during the dark period and reducing the amount of water given to each plant from 50 to 30 ml/day. In the second experiment with the cultivar Diamant, 30 plants were used of which 16 were monitored by CT analysis. After tuber initiation, as determined by CT monitoring, drought and mild heat stresses were applied to half of the plants for 2 weeks while the other half of the plants served as control group. The control group was watered with 50 ml per day/plant for the whole time while the stress group was subjected to the conditions described above.S-Medium  containiAll plants were measured at an individually designed CT system at the Fraunhofer EZRT in Fuerth, Germany using a GE 225 MM2/HP source, Aerotech axis systems and the Meomed XEye 2020 Detector operating with a binned rectangular pixel size of 100 μm see . The souWe generated an automated data analysis platform for the high-throughput CT analysis. For better performance the image processing was done in a special C++ application which can be used in the VolumePlayerPlus visualization and segmentation software. To analyze all plants in a comparable manner, special segmentation algorithms were used. Only using the reconstructed linear absorption coefficient, results in many false positives and it would be very hard to distinguish tubers from different plant material inside the soil . Thus, we used the hypothesis that the soil is inhomogeneous compared to tubers for our image segmentation pipeline which contains eight steps see .As first step after the reconstruction we applied a size adaptable median filter in 3D to reduce the noise. On this filtered volume we calculated pixel wise the local variance:Now, compared to the soil, tubers are more homogeneous and a single threshold binarization separates most of the surrounding soil from the tubers. However, in this state potato tubers and stolons are still connected. Therefore, we applied a morphological operator called Erosion . As nextAt this stage only homogenously labeled objects were left over in the reconstructed dataset. For all of these objects, parameters like the volume, the mean absorption coefficient, the position and the aspect ratio were calculated. Scanning the same pot several times in a row, allowed to further differentiate between tubers and other false positive segmented homogenous objects like stones and parts of unconnected stolons. A tuber has to grow over time and the mean reconstructed absorption coefficient is depending on the material and density , thus, iUsing these algorithms, it is possible to reconstruct, segment and analyze all plants at each measured time point with only one set of parameters. Both experiments combined 592 measurements in a total of 36 plants. An evaluation version of the Tuber segmentation package as well as some reference volumes can be obtained by the senior author (SG).via CT imaging were harvested and biomass was determined. At each time point and condition, 10 tubers were selected for sampling. Samples were immediately frozen in liquid nitrogen and stored at −80°C until further use.Tuber samples from the experiment with the cultivar Diamant were taken at three time points; (1) 8 days after initialization of the stress period, when the tubers had stopped growing (TP1), (2) 3 days after the stress period, when the tubers had started growing again (TP2), and (3) at the end of the experimental period, 2 weeks after the end of the stress phase (TP3). At each time point five plants per treatment were harvested and the leaf and tuber biomass were measured with a laboratory balance. At the first time-point, 10 plants (five per condition) were harvested which had not been monitored by CT analysis. At the second time-point, two plants per condition had not been subjected to CT analysis, while the other three sampled plants had been monitored. At the end of the experimental period the remaining 10 plants which had been monitored Starch was quantified in tuber samples extracted with 80% ethanol incubated for 1 h at 80°C. After centrifugation, removal of the supernatant, and washing with ethanol and water, 0.2 M Hepes-KOH, pH 7.5 was added and samples kept at 4°C overnight. After heating the samples to 95°C for 1.5 h, the pH was neutralized with 1 N acetic acid. Next, starch extracts were incubated with Amyloglucosidase overnight at 55°C and pH 5.5. The spectrophotometric determination of released glucose was conducted as described previously .Sucrose Synthase activity was measured according to RNA was isolated as described previously . Total RTM H minus first strand copyDNA (cDNA) synthesis kit (Thermo Scientific) according to the manufacturer’s instructions. For relative quantification of starch gene derived transcripts, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses were performed using the AriaMX qPCR system (Agilent Technologies) in combination with the Brilliant II SYBR® Green QPCR Master Mix (Agilent Technologies) with four biological replicates for each condition and two technical replicates. Ubi3  express[L22576,  to have 2 of 0.99.Analyzing the volume and the mean linear absorption coefficient for each tuber allows to calculate a virtual fresh weight. For this assumption we used the high correlation between the linear absorption coefficients of the reconstruction of the 3D volume with the physical density of the object. However, this only holds, as long as the elemental composition of the object of interest is more or less constant. In case of potato tubers, the elemental composition is mainly determined by hydrocarbon chains and water. In Applying this as calibration, it was possible to directly calculate the corresponding fresh weight during the growth process for each measured tuber. With the results of the depicted image processing pipeline and the corresponding calculated features, a python script collected each single result and calculated the growth statistics over time for each potato tuber and variety.via CT imaging every second day. After the measurement the potato tubers were virtually excavated out of the soil for each time point. This segmentation process was automated and ran with the same parameter for all plants even if there were differences in potato tuber sizes or in soil moisture due to stress conditions. Potato plants were monitored During the first experiment, five different genotypes were measured every second day from day 14 after planting until day 42 after planting. From day 15 after planting until day 29 after planting combined drought and heat stress was applied. The plants had between three and nine tubers which shows that the algorithm can segment the tubers even if the space between the tubers is restricted. In Additionally, the total tuber biomass was calculated for each plant individually as the sum of all tuber weights of the respective plant at each time-point and the average total tuber fresh weight was calculated for all plants of the same cultivar. For all genotypes analyzed, the increase of total tuber biomass stagnated during the combined abiotic stress treatment between days 15 and only very small increments in tuber biomass were observable . After tVia the CT analysis pipeline, we monitored the tuber growth three times per week in 16 pots and utilized the same calibration set , 3 days after cessation of stress treatment (TP2) and after a 14-day recovery phase at the end of the experimental period (TP3) were subjected to qRT-PCR analysis. Stress-responsive genes were selected from publications on drought stress and subsequent re-watering in potato stolons , potato DI19), Abscisic acid and environmental stress-inducible protein (TAS14), Lipoxygenase (LOX1), and Heat-shock Protein (HSP) was significantly increased during the heat treatment at TP1 when compared to potato tubers grown under control conditions  a marked decrease in SuSy4 expression in comparison to the control group was detected  SuSy4 expression in stress treated tubers exceeded the expression in control tubers GBSS) was analyzed. Similar to SuSy4, GBSS expression was found to be significantly down-regulated in tubers of the stressed group during the stress treatment (TP1) and to recover to control values after the stress , GPT2.1 expression was slightly, but non-significantly higher in tubers grown under stress conditions than in tubers grown under control conditions  and automated way (one set of parameter for the whole experiment). This enabled analyzing the stress dependend growth dynamic of individual tubers within one pot and to visualize even small variations in belowground biomass. The time dependance of this data set allowed the 4D analysis of growth processes affected by abiotic stresses. The imaging pipeline presented here can be scaled up to be used in high-throughput phenotyping systems. However, the combination with automated data processing is the key to generate objective data accelerating breeding efforts to improve abiotic stress tolerance.We chose combined heat and drought stress since these abiotic stresses are likely to occur in parallel in the context of global climate change . We applDue to the early timing of the stress treatment directly after tuber induction, it seems that most tubers were able to recover from the implications of the stress treatment on biomass accumulation. This was also seen in an early study on the effects of individual heat and drought stress on tuber development  where eaTAS14 was 4.7-fold up-regulated after 3 days of drought treatment and 8.2-fold down-regulated after re-watering when compared to stolons from plants grown under control conditions. In our study, TAS14 expression in tubers was almost 50-fold up-regulated after 8 days of combined drought and heat stress but had returned to values similar to control 3 days after stress release. This indicated that TAS14 might be suitable as a marker for stress in potato tubers. However, further characterization of its expression profile in different plant organs and under differing conditions is needed to confirm its suitability as a stress marker. Support for the role of TAS14 during abiotic stress comes from experiments in tomato, where stable overexpression of TAS14 led to improved long-term drought tolerance  during elevated temperature. The strong induction which we have determined for HSP could be a result of the additional drought treatment and the different methodology . Thus, HSP might be an appropriate marker for combined heat and drought stress in potato tubers but further validation is recommended.Heat-shock protein (DMT400032851) which was strongly elevated in potato tubers during combined heat and drought stress compared to control, has previously been found in a microarray analysis among 2,886 differentially expressed genes in potato tubers of the cultivar Desirée during mild heat . In the SuSy4 as well as specific activity of SuSy, a marker for starch biosynthesis, in tuber samples. Increased SuSy expression and activity has been associated with increased starch and total yield (SuSy4 expression and SuSy activity have been shown decrease (Heat and drought are abiotic stress factors influencing many developmental and physiological processes. In potato plants, both factors, alone or in combination, affect tuberization and starch accumulation associated therewith . Dependial yield . Under adecrease .SuSy4 expression (GPT2.1 expression would decrease during stress treatment. However, gene expression analysis of GPT2.1 revealed no significant differences between stressed tuber samples and tubers grown under control conditions. This is consistent with the gene expression data from tuber samples under elevated temperatures published by GPT2.1 was not among the differentially regulated genes.GPT2.1 has been identified as the tuber-specific GPT2 isoform , whose epression . TherefoGBSS was found to be significantly down-regulated in potato tuber during combined heat and drought stress in our qRT-PCR analysis. Similarly, in the microarray experiment by GBSS expression was downregulated significantly in the tuber samples from plants grown under elevated temperature. This seems consistent with an overall decrease of starch biosynthesis in potato tubers under heat and drought stress. In our experiment, SuSy4 and GBSS expression recovered to levels of tubers grown under control conditions after the stress conditions were released. Similar observations were reported by Granule-bound starch synthase is the starch synthase isoform responsible for amylose-synthesis . Expressin planta and enables also methods like guided sampling to correlate in future morphological and physiological measurements. This is in accordance with for example The presented imaging pipeline was able to generate stable results in growth analysis of potato tubers for five different genotypes. Using only one set of parameters it was possible to track individual tubers for different growth stages and a high variation in tuber number per plant. Thus, we demonstrated that this kind of CT analysis is a suitable tool to monitor morphological changes of otherwise inaccessible, underground tissues In general, the throughput of the system has to be evaluated in terms of cycle time in the measurement—including the measurement time and the time for sample exchange—and the time for data processing. The data processing time is directly connected to the computing power used for the analysis and with the data analysis pipeline presented a parallel computing can be realized. Thus, the processing time is not a bottleneck to further increase the overall throughput of the combined system. However, the current system was relying on a manual change of the individual plants. To further increase the cycling time of the system an integration in a conveyor system would be highly beneficial. Doing so, the time for sample exchange could be minimized. To decrease the measurement further, there are some more steps possible. The easiest one is to decrease the resolution and at the same time increase the smallest detectable tuber. This option is strongly connected with the breeding use case in mind. If this is not an option, using stronger X-ray sources or even pulsed sources is a way to decrease the scanning time. Finally, a vertical gantry system with pulsed X-ray sources with rotating anodes would be a possibility for real high-throughput tuber analysis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JC, SG, and JV performed the experiments and drafted the manuscript. JV did the molecular biological work. SG supervised and together with JC performed the CT work. NW and JC performed the image and data analysis. US and NU provided the project funding, conceived and led the study, and contributed to writing the manuscript. US, SG, and JL discussed the work and the biphasic growth pattern. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the characteristics of treatment-naïve nonexudative macular neovascularization (MNV) in age-related macular degeneration before the onset of exudation using swept-source optical coherence tomography angiography.MNV area, choriocapillaris (CC) flow deficits (FDs), vessel area density (VAD), vessel skeleton density (VSD), retinal pigment epithelial detachment (PED) volume, mean choroidal thickness (MCT), and choroid vascularity index (CVI) measurements were assessed at two visits prior to exudation. We compared measurements made at the second visit and the rate of change between visits in eyes with and without exudation. The differences in these parameters between eyes with and without subsequent exudation were summarized with 95% confidence intervals (CIs)..Twenty-one eyes with nonexudative MNV were identified and followed. Nine eyes developed exudation, and 12 eyes did not develop exudation. Differences between these groups of eyes for all parameters tended to be small, and the 95% CIs largely ruled out any substantial differences. Overall, eyes with exudation had 24% smaller VAD, 20% smaller VSD, and 33% smaller PED volume measurements. No noteworthy differences were observed for MNV area, CC FDs, MCT, or CVI measurementsThe onset of exudation was correlated with lesions having less vascularity and smaller PED volume measurements, but measurements of MNV area, CC FDs, MCT, and CVI were not correlated with near-term exudation. Investigations are ongoing to further explore these and other anatomic changes as harbingers of near-term exudation. The institutional review board of the University of Miami Miller School of Medicine approved the study, and all patients signed an informed consent for this prospective OCT study. The study was performed in accordance with the tenets of the Declaration of Helsinki and complied with the Health Insurance Portability and Accountability Act of 1996.2) were excluded due to the difficulty of measuring these lesions reproducibly. Only lesions fully contained within a 6 × 6-mm scan area were included in this study due to the need for reproducible measurements under and around the lesions. In addition, eyes with geographic atrophy (GA) were excluded due to the difficulty of quantifying the CC within the area of GA. The patients were divided into two groups based on whether the nonexudative MNV developed exudation. If the nonexudative MNV developed exudation, then the last two visits before the visit when exudation was documented were selected for analysis. If the MNV did not develop exudation, then the two consecutive visits at least 6 months before their last follow-up visit were used for analysis to ensure a buffer between the analysis date and possible future exudations. Time of symptomatic exudation in this study was defined as the day the patient received anti-VEGF treatment. The presence of exudation was determined by reviewing individual B-scans to detect intraretinal or subretinal fluid. In this study, the accumulation of fluid under the retinal pigment epithelium (RPE) in the absence of subretinal or intraretinal fluid was not considered evidence of exudation, and anti-VEGF therapy was not offered. The volumes of these PEDs were assessed as described below.Between April 2016 and June 2019, patients with AMD with treatment-naïve, nonexudative MNV were identified and underwent SS-OCTA imaging by the same technician during their follow-up visits. Follow-up visits were determined by routine clinical care and not dictated by a formal protocol. Some patients were followed for bilateral nonexudative AMD, and other patients were seen more frequently due to the need for intravitreal injections in their fellow eyes. The last follow-up visit included in this study was February 10, 2020. Patients were seen usually every 2 to 3 months. Only eyes with subclinical type 1 MNV were included. Eyes with subclinical type 3 MNV and polypoidal choroidal vasculopathy were excluded. Eyes with small neovascular lesions (lesion size at visit 1 < 250 µm in greatest linear dimension or 0.2 mmc) algorithm as previously described.–SS-OCTA images were acquired using an instrument  with a swept-source laser source that had a central wavelength of 1050 nm and a scanning speed of 100,000 A-scans per second. The axial resolution of the system is ∼5 µm in tissue, and the lateral resolution is ∼20 µm at the retinal surface. Both 6 × 6-mm and 12 × 12-mm scan patterns were acquired for each patient at all visits. All scans were centered on the fovea and were acquired using FastTrack motion correction software. OCTA images on the PLEX Elite 9000 are generated using the complex optical microangiographic (OMAGThe 6 × 6-mm scans were used to compute the CC FD percentage (FD%) and the average CC FD area measurements around the MNV. Visualization and quantification of the CC were obtained using a 16-µm thick slab with its anterior boundary located 4 µm beneath the Bruch's membrane (BM).To quantify the vascularity of the MNV, VAD and VSD were measured from the 6 × 6-mm scans. Because the RPE complex is highly light scattering and reduces the quality of the OCTA MNV images for detailed vascular quantification, we specifically modified our previously validated retinal vascular quantification algorithmThe volumetric measurements of the PEDs were obtained from 6 × 6-mm scans. The legacy RPE segmentation and the BM segmentation (from the multilayer segmentation as provided by the instrument automated segmentation software) were edited on the instrument as needed to ensure appropriate boundary segmentation. The processed OCT datasets were exported from the instruments and uploaded to the Advanced Retina Imaging Network on the Advanced Retina Imaging Network Hub provided by Carl Zeiss Meditec, Inc. The PED volume was calculated using the Advanced RPE analysis v0.9 algorithm. This algorithm uses the edited RPE and BM segmentations from the instrument as the two segmentation boundaries to measure the PED area and volume, and an en face color-coded PED volume map was created, as well. Because some RPE elevations were associated with drusen while others corresponded to the double-layer sign associated with the MNV, the MNV masks generated by using the ORCC slab as previously described above were superimposed on the PED volume maps in order to isolate the PED volume that was specific for the MNV, which is referred to as MNV–PED volume. Square-root transformation of area measurements and cube-root transformation of volume measurements were performed to eliminate the impact of lesion size on the variability of the standard deviation previously determined from test–retest measurements. The advantages provided by using these transformations were previously described.Mean choroidal thickness (MCT) and CVI measurements were obtained from the 12 × 12-mm structural SS-OCT scans using an automated strategy as previously reported by Zhou et al.t-test 95% confidence intervals (CIs) around the differences between eyes that did and did not exhibit exudation after visit 2. These 95% CIs allow us to draw conclusions about the likely size of these differences even with relatively small sample sizes. The normality assumption of the t-test was assessed with the Shapiro–Wilk test, and for measurements that violated this assumption, we estimated 95% CIs with bootstrap methods using 2000 resamplings per interval.,Statistical analyses were performed using SPSS Statistics 25 . Data were summarized with mean, standard deviation (SD), median, and interquartile range (IQR). In this descriptive study of many different measurements, we limited statistical inference to the calculation of two-sample 2); two eyes had neovascular lesions that extended outside the 6 × 6-mm scan area; and six eyes from five patients had macular pathology other than AMD, including multifocal choroiditis, pathological myopia, and a choroidal nevus.A total of 72 eyes from 70 patients with nonexudative MNV were identified using SS-OCTA imaging between April 2016 and June 2019. Among these 72 eyes, 51 eyes from 50 patients were excluded for further analysis for the following reasons: 14 eyes had inadequate follow-up; 14 eyes had GA at the baseline visit; four eyes developed exudation without having completed two prior visits; five eyes had type 3 MNV; one eye had a polypoidal choroidal vasculopathy lesion; five eyes had small neovascular lesions that were difficult to measure in a reproducible manner . In the eyes that did not develop exudation, the last two visits were at least 6 months before the cut-off for this study, with the average interval from visit 2 to the latest nonexudative status check being 14.8 ± 6.5 months . For the group that did develop exudation, the average interval from visit 2 to the day of exudation was 2.1 ± 1.3 months . Six out of 18 (33%) monofocal eyes developed exudation compared to three out of three (100%) multifocal eyes, a difference that did not reach statistical significance . Of the three multifocal eyes, one eye had two foci and two eyes had three foci.A total of 21 eyes from 20 patients with nonexudative MNV secondary to AMD were followed in this study. Twelve eyes from 11 patients did not develop exudation, whereas nine eyes from nine patients developed exudation. For eyes that did not develop exudation, the last two visits (defined as visit 1 and visit 2) at least 6 months prior to the last follow-up were selected for assessment. The average time between visit 1 and visit 2 was 3.6 ± 2.1 months. For eyes that developed exudation, the last two visits (visit 1 and visit 2) before exudation were selected for assessment, with the average time between these two visits being 2.7 ± 1.7 months. The difference in the intervals between the last two visits for each group was not statistically significant  were not associated with exudation. However, our attention remains focused on the perfusion of the CC, and this parameter must be studied in a larger sample of patients, as the change in R1 CC FD% was associated with an asymmetric 95% CI. This outcome was of interest because we hypothesized that there would be an increase in CC FD% immediately adjacent to the MNV prior to the onset of exudation or that there would be a difference in CC FD% in the short term when comparing neovascular lesions that remained dry versus lesions that developed exudation. The hypothesis that a difference in CC FD% would exist stems from the current consensus among researchers that MNV arises as a compensatory response to underlying CC impairment, and the ingrowth of MNV is an attempt by the eye to recapitulate the CC in response to growth factors released by distressed RPE and photoreceptors.Other surrogates that might reflect changes in the CC, such as changes in the underlying choroidal vasculature, were explored by measuring the MCT and the CVI, but these were not associated with the onset of exudation in our current study. The rationale for a relationship between the risk of exudation and the mean CT and CVI measurements comes from the expected correlations between these two parameters and choroidal blood flow, as both measurements are directly related to the choroidal vascular volume as previously reported by Zhou et al.Our findings were different from the results of Teo et al.,,,Contrary to expectations, our data suggest that exudation generally developed in eyes with decreased VAD and VSD measurements and in eyes with smaller MNV–PED volume measurements. Although our findings must be confirmed, it is intriguing to speculate that, if true, then it might reflect a scenario in which the smaller MNV cannot adequately provide the needed nutritional support for the overlying RPE and photoreceptors in the setting of compromised CC perfusion, and the nutritionally deprived RPE and photoreceptors respond by overproducing VEGF. Interestingly, Seddon et al.Because the sample size of this study was small, we calculated 95% CIs around the differences at visit 2 and the change rates between eyes that did or did not go on to exudation to assess the likely size of differences. Review of these 95% CIs suggests that, despite the small sample size in this study, we can rule out a sizeable difference in the rates of change between eyes with exudation and those without exudation, except that there is a possible faster increase in CC R1 FD% in eyes with exudation. We cannot rule out moderate differences in measurements made at visit 2 between the two groups, although MNV size is unlikely to be larger in the eyes with exudation compared with those without exudation. Although our VAD, VSD, MNV–PED volume, and CC R1 FD% measurements are of interest, we must emphasize that, for all of the parameters studied in this current investigation, our results must be interpreted with caution until they are validated in future studies.Interestingly, three out of nine eyes that developed exudation had multifocal MNV lesions, whereas none of the eyes that stayed dry had multifocal MNV lesions. That there was no significant difference in the number of lesions between groups suggests that eyes with multifocal MNV may have to be monitored more closely due to the increased risk of exudation. Whether this increased risk of exudation is based on just the increased number of lesions or an increased underlying susceptibility to develop exudation remains to be determined.P values. We view our results as exploratory and will only be persuaded of their robustness when reproduced with a larger dataset. We remain intrigued with the CC FD% within R1 region. However, even if this region proves to be a useful predictor of exudation in a larger population, it is unlikely that, when measured during the routine follow-up of patients with nonexudative MNV, this CC FD% measurement within R1 would prove to be useful as a definitive near-term predictive metric of exudation at the patient level given the need for a large number of patients to detect this significance, if it exists. Another limitation of our study is the lack of monthly or weekly follow-up visits in the protocol that might have detected changes in the nonexudative MNV that would have occurred closer to the onset of exudation. In our current ongoing prospective natural history study of AMD patients with intermediate AMD or GA, patients are being followed at fixed follow-up intervals of every 3 months; however, this 3-month interval still will not be sufficient to explore whether very near-term changes are correlated with the onset of exudation, but this dataset will allow us to validate the results presented in this report. Although more frequent follow-up visits will be needed to determine if changes might occur within days or weeks prior to exudation, the feasibility of running such a natural history study in an elderly population with AMD is challenging. Our current experience with a schedule of visits every 3 months suggests that most patients would not be willing to return more frequently when no treatment is being offered. With the development of home OCT monitoring, it might be possible to acquire some of the near-term data by remote monitoring. Finally, in order to properly quantify the CC FDs, only 6 × 6-mm scans with a scan density that provided 12-µm spacing between A-scans and B-scans were used for accurate quantitation of FDs. As SS-OCTA scanning speeds increase, we should be able to acquire larger scans with higher scan densities, which will allow us to study CC FDs surrounding larger nonexudative neovascular lesions in the future.The major limitation of our study is its small sample size. Based on this small sample size and the numerous statistical comparisons that we have performed, we present these data as a descriptive study and have refrained from reporting In summary, we used SS-OCTA imaging to follow eyes with treatment-naïve nonexudative MNV, and we measured various parameters including lesion size, CC FD%, VAD, VSD, MNV–PED volume, MCT, and CVI and compared these measurements between eyes that did or did not develop exudation. Although the eyes that developed exudation seemed to have lower VAD and VSD measurements, smaller MNV–PED volume measurements, and a decrease in VSD over the visits prior to exudation, we interpret these results with caution. The search will continue for characteristic anatomic and blood flow measurements in eyes with nonexudative neovascular lesions that may be useful to predict near-term exudation. Until these markers of near-term exudation are identified and validated, home monitoring and close clinical follow-up are recommended so that anti-VEGF therapy can commence as soon as symptomatic exudation develops.


To evaluate the ability of the NEWS2-L  scale to predict the risk of early clinical deterioration  in patients with dyspnoea treated by the Medical Emergency Services compared with NEWS2 and lactate in isolation.Prospective, multi-centre study of a cohort of 638 patients with dyspnoea treated in the ambulance and priority-transferred to a hospital emergency service in the cities of Valladolid, Salamanca, Segovia or Burgos (Spain). We collected clinical, analytical and demographic data. The main outcome measure was all-cause mortality within 48 hours. The recommendations of the Royal College of Physicians were followed to calculate NEWS2. When NEWS2 and LA prehospital values were obtained, the two values were added together to obtain the NEWS2-L.p<0.001, with a significant decrease between the value at 48 hours and at 30 days.Mortality within 48 hours was fifty-six patients (8.8%). The NEWS2-L scale obtained an area under the curve (AUC) of the receiver operating characteristics (ROC) for mortality within 48 hours of 0.854 (CI 95% 0.790-0.917), at seven days of 0.788 (CI 95% 0.729-0.848) and at 30 days of 0.744 (CI 95% 0.692-0.796); in all cases The NEWS2-L scale was found to be significantly superior to the NEWS2 scale and similar to lactate in predicting early clinical deterioration in patients with dyspnoea. This scale can help a nurse detect these patients early, as part of their regular practice, and thus guide therapeutic efforts. Dyspnoea is defined as a subjective feeling of a lack of air or difficulty breathing and is a symptom present in a great variety of pathologies, often associated with different degrees of respiratory failure. DyspnoeaAt the prehospital level, it is a common reason to seek medical care, with different levels of mortality and morbidity.-10) The role of lactate (LA) as a predictor of poor prognosis generally in patients treated by mobile and hospital emergency services is also well known, is one of the scales validated in the prehospital setting and has a correlation between a high prehospital score in NEWS2 and a higher incidence of adverse outcomes.8ll known, acting all known,The detection and handling of early signs of deterioration in the patient is a highly complex process influenced by many factors. In this regard, nursing personnel play a very important role in identifying clinical deterioration in potentially critical patients due to how closely they work with the patient. Understanding and handling early warning scales like NEWS2 and the meaning of biomarkers like LA is particularly important in these circumstances and can serve as an aid for nurses to guide their actions. The aim Study design and ethical considerations. We conducted a prospective, multi-centre cohort study on an opportunity sample of patients with dyspnoea treated by an Emergency Mobile Unit (EMU) in the cities of Valladolid, Salamanca, Segovia and Burgos (Spain). These patients were subsequently transferred to their reference hospital. The main outcome measure was all-cause mortality within 48 hours. Secondarily, mortality at seven and 30 days after treatment by the EMU was studied at a global level.Participants. Between 1 April 2018 and 30 June 2019, patients aged ≥ 18 years were included if they had called the 112 emergency number requesting urgent help and after assessment by the ambulance (EMU) on the scene were determined to have dyspnoea  as the reason for their call and were taken to their reference hospital by ambulance. Excluded from the study were patients where it was not possible to obtain informed consent, patients under the age of 18, pregnant women, patients with an acute psychiatric disorder or documented terminal disease, patients who died during the callout or transfer, patients evacuated by other means of transport  or patients who following assessment by the EMU required no further urgent care and were discharged onsite.Variables and data collection. The endpoints and predictors were collected by independent researchers at each hospital, obtained from a review of patient electronic medical records. The primary endpoint was collected by the clinical investigators tasked with data collection. Epidemiological values were also collected during the callout , as were the clinical values needed to calculate NEWS2 . Analytical values (glucose and venous LA) were also collected. The prehospital main diagnosis was also recorded on the basis of the International Classification of Diseases . The heart rate, blood pressure and oxygen saturation measurements were performed with the LifePak® 15 monitor . Temperature was obtained with a tympanic thermometer, model ThermoScan® PRO 6000 , glucose was obtained with the FreeStyleOptium Neo glucometer  and LA with the Accutrend Plus lactometer .By reviewing the patient’s electronic medical record 30 days after the index event, the following hospital values were recorded: admission, need for ICU, in-patient days and mortality at 48 hours and at seven and 30 days.NEWS2-L determination. The recommendations of the Royal College of Physicians were folScale 2 should be used in patients with hypercapnic respiratory failure to weigh up the oxygen saturation score. Each category was classified from zero to three points. The scores of each category were added together to obtain a total. Composite scores over five (or three in any of the parameters) triggered an urgent review. A score over seven triggered a review by the critical care team or an advanced medical response team. Abbreviations: NEWS2: National early warning score 2; HR: heart rate; RR: respiratory rate; T: temperature; SBP: systolic blood pressure; SpO2: oxygen saturation; suppl. O2: supplemental oxygen; GCS: Glasgow coma scale. Taken from the Royal College of Physicians.The LA values were collected by the nursing personnel of each EMU in three phases: 1) The test strip was inserted after switching on the instrument. 2) A drop of venous blood (15-40 μL) was deposited on the test strip. 3) The lid was closed and a result obtained after 60 seconds. The maximum time between blood collection and placement of the sample in the device was one minute. All the measuring devices were calibrated every 100 measurements, always by the same researcher, using the Accutrend® BM-Control-Lactate control solution . When the NEWS2 and LA prehospital values were obtained, the two values were added together to obtain NEWS2-L.Statistical analysis. All the data was stored in an XLSTAT® BioMED database for Microsoft Excel® version 14.4.0. , and SPSS version 20.0.  which were also used for statistical analysis. The database was cleaned prior to statistical analysis by means of logical tests, range tests  and data consistency. The presence and distribution of unknown values of all variables were subsequently analysed. Qualitative variables were described by absolute frequencies with their 95% confidence interval (CI 95%). Quantitative variables were described as median and interquartile range (IQR). The Mann-Whitney U-test was used to compare quantitative variables. The Chi-square test for two-way tables or the contrast of proportions was used to determine the association or dependence relationship between qualitative variables; if necessary , we used Fisher’s exact test. NEWS2, NEWS2-L and LA area under the curve (AUC) of the receiver operating characteristic (ROC) was calculated for mortality at 48 hours and at seven and 30 days, along with the best score in each case for greatest combined sensitivity and specificity (Youden index). We also calculated the positive predictive value (PPV), negative predictive value (NPV), positive probability ratio (PPR) and negative probability ratio (NPR) for these scores. In all tests, a confidence level of 95% and a p-value of less than 0.05 were considered significant. The values obtained were subsequently compared against each other, establishing a comparison between the three tests studied (NEWS2-L vs NEWS2 vs LA).Ethical aspects. The study was approved by the Clinical Research Ethics Committees of all the participating centres. All the patients (or guardians) signed the informed consent form. The highest safety standards were followed at all times, protecting participant confidentiality and complying with the national and international regulations included in the Declaration of Helsinki on biomedical research involving human subjects.A total of 638 patients with dyspnoea were included between 1 April 2018 and 30 October 2019 . MortaliStratifying the sample by predominant pathology in patients with dyspnoea who were treated and transferred, 170 (26.6%) were patients with chronic obstructive pulmonary disease (COPD), 164 (25.7%) were patients with heart failure (HF), 198 (31%) were patients with infections and the remaining 106 (16.6%) were patients with any condition other than the above, covered under the mixed group of ‘Other’. The values of the patients’ clinical/epidemiological characteristics were provided as absolute and percentage values or as median and interquartile range as appropriate. Abbreviations: IQR: interquartile range; RR: respiratory rate; SpO2: oxygen saturation; O2: oxygen; SBP: systolic blood pressure; HR: heart rate; GCS: Glasgow coma scale; LA: lactate; NEWS2: National Early Warning Score 2; NEWS2-L: National Early Warning Score 2 Lactate; COPD: chronic obstructive pulmonary disease; HF: heart failure; ICU: intensive care unit .Comparing the NEWS2 scale vs LA vs NEWS2-L, the NEWS2-L scale obtained the best AUC-ROC for mortality at 48 hours with 0.854  with a cut-off point of 12.2. Similarly, the NEWS2-L scale exceeded the other two tests for seven and 30 days, with AUC-ROCs for the NEWS2-L scale of 0.788  and 0.744  and cut-off points of 12.7 and 12.3, respectively. Comparing the three tests, lactate obtained the best sensitivity (91.1%) and specificity (79.4%) in mortality at 48 hours, followed by the NEWS2-L scale and with a considerable difference with respect to the NEWS2 scale .In view of the results obtained, we can see that the NEWS2-L scale has a good ability to predict early clinical deterioration in patients with dyspnoea, significantly superior to the NEWS2 scale and similar to LA.We also found that both analysed scales and LA lost their predictive capability over the long term. This loss of efficacy was most pronounced in the case of LA, which might be related to the fact that this biomarker increases considerably in situations of hypoxia and hypoperfusion and thatet al. , a superiority that was maintained at one, two, three and four weeks. These findings are in line with our own findings between the NEWS2-L vs NEWS2 scales, although we evaluated mortality at 48 hours and at seven and 30 days. Another study by Young et al.. It produced similar data in some aspects and an AUC-ROC of 0.91 (CI 95% 0.83-0.86) for mortality at 48 hours. However, that study referred specifically to the prehospital setting and included patients with all types of conditions, unlike the present study that only included patients with dyspnoea, so the obtained results are not directly comparable, although they do shore up the aforementioned idea of how associating LA with an early warning scale can improve its performance.et al., in their systematic review(Even though it is obvious that no scale can replace a suitable patient history and clinical examination by highly qualified personnel, many studies have concluded that the different early warning scales are a useful and efficient tool in predicting clinical deterioration among potentially serious patients, enabling a better clinical approach.-24 Karloic review analysinThis study has a number of limitations: firstly, there are many early warning systems, making it hard to select between them, although, as shown throughout the study, the NEWS2 scale has very high psychometric values today and is extensively used in both hospital and prehospital settings. Secondly, LA was used for its demonstrated clinical utility, but there are other analytical parameters such as blood gases, electrolytes, etc., which can today be obtained in the prehospital setting and could offer more data for clinical patient handling. Finally, this study focused on patients whose reason for assistance was dyspnoea and studied their mortality at 48 hours and at seven and 30 days after the values obtained at the time of prehospital care, but going forwards it would be interesting to have studies that assess the way the scale evolves using serial measurements in the hospital setting and appraising their efficacy in other conditions. It could also be very interesting to obtain and compare several early warning scales against each other and obtain other analytical values at the highest levels of lactate, studying the combinations that offer the most efficacy for predicting early clinical deterioration by groups of patients with different conditions.In short, and after analysing the AUC-ROCs mentioned previously in the results, we can state that the NEWS2-L scale performed better than the NEWS2 scale and similar to LA in predicting mortality within the first 48 hours in patients seen for dyspnoea, and that the NEWS2-L scale beat LA as a predictor of mortality at seven and 30 days and was therefore superior overall. This scale can be useful in detecting patients likely to suffer significant clinical deterioration and more mortality from the time of their prehospital treatment, helping guide health professionals’ efforts to choose more or less intensive therapies with the aim of safeguarding the patient. Its regular use by emergency medical services could be considered to develop the most efficient and suitable response.In the specific case of nurses, their role is fundamental to the correct implementation of the scale since they are the people who regularly collect the data needed to prepare it. Having a validated, well-performing scale like this facilitates their regular practice in the early detection of patients who are starting to deteriorate and can increase and guide therapeutic efforts. Correctly collecting the necessary variables, applying the scale and interpreting the result obtained, then consequently acting on it are also a challenge for nurses.


Endothelial and epithelial cellular barriers play a vital role in the selective transport of solutes and other molecules. The properties and function of these barriers are often affected in case of inflammation and disease. Modelling cellular barriers in vitro can greatly facilitate studies of inflammation, disease mechanisms and progression, and in addition, can be exploited for drug screening and discovery. Here, we report on a parallelizable microfluidic platform in a multiwell plate format with ten independent cell culture chambers to support the modelling of cellular barriers co-cultured with 3D tumor spheroids. The microfluidic platform was fabricated by microinjection molding. Electrodes integrated into the chip in combination with a FT-impedance measurement system enabled transepithelial/transendothelial electrical resistance (TEER) measurements to rapidly assess real-time barrier tightness. The fluidic layout supports the tubeless and parallelized operation of up to ten distinct cultures under continuous unidirectional flow/perfusion. The capabilities of the system were demonstrated with a co-culture of 3D tumor spheroids and cellular barriers showing the growth and interaction of HT29 spheroids with a cellular barrier of MDCK cells.  Cells lining the vasculature form barriers separating blood from its surrounding tissues and play a vital role in the selective transport of solutes and other molecules across these barriers. Endothelial cells that constitute the lumen of intact blood vessels form a continuous monolayer that acts as a barrier between blood and the surrounding tissues ,2. EpithIn many cases of chronic inflammation and disease, the integrity of the cellular barriers protecting vital organs of the human body tends to be disturbed. For instance, persistent neuroinflammation that has been reported in the pathology of almost all neurodegenerative disorders is accompanied by a disruption in the BBB integrity ,5. In adTo enable efficient drug screening and toxicity studies, in vitro models of cellular barriers must exhibit the characteristics and properties seen in vivo, such as the expression of junctional proteins and complexes. Transwell-based platforms are most often used to model cellular barriers in vitro; however, they lack critical properties such as the shear forces associated with physiological blood flow ,28. As aWith the advent of microfluidic organ-on-chip technology, it has become easier to model miniaturized healthy and diseased human tissues in vitro with increased physiological relevance. Advancement in microfabrication, microfluidics, biomaterials and tissue engineering techniques have been exploited to establish relevant test systems via the co-culture of multiple cell types, with cells often embedded in a hydrogel, to recapitulate the 3D spatial organization, complexity and heterogeneity of in vivo tissues ,35,36. ATherefore, we exploited microfluidic technology to model cellular barriers in a parallelizable microfluidic platform manufactured by microinjection molding of cyclic olefin copolymer (COC) comprising integrated electrodes to enable real-time transepithelial/transendothelial electrical resistance (TEER) measurement.Continuous unidirectional flow was achieved by means of gravitation-driven flow in combination with an incubator compatible perfusion system. TEER was measured by employing a custom-made parallelized fast Fourier transform (FFT)-impedance spectrometer. As an example, and to demonstrate the versatility of this device, we established the complete workflow ranging from cell seeding to the generation of co-cultures of tumor cells growing in a hydrogel mimicking tumor extracellular matrix (ECM) along with a cellular barrier. Here, we show preliminary data to demonstrate how this system can be used to monitor the growth of micro-tumors and their interaction with a cellular barrier and migration into the perfusion channel.The microfluidic chip (MFC) was manufactured from cyclic olefin copolymer (COC) by microfluidic ChipShop GmbH, Jena, Germany. COC is transparent and exhibits low autofluorescence. It comprises ten micro channels with five different layouts of pillar size and gap width . The molThe chip body was manufactured by precision injection molding. The channel depth was 170 µm. The process yields devices with excellent optical properties and low substance adsorption ; the latFinally, microfluidic structures, electrodes and connecting leads were covered by bonding a thin (approx. 140 µm) COC foil onto the bottom of the chip, leaving connecting pads open to enable electric connection to the electrodes.The described fabrication process is scalable in regard to numbers and yields devices in reproducible quality.www.cellendes.com) before filling the microfluidic chip. The pre-gel solution mixture was filled into the abluminal side of the micro channels with the help of a syringe pump under a constant flow rate of 2.5 µL/min  before the microfluidic chip was filled as shown in The reagents were obtained from Cellendes GmbH, Reutlingen, and mixed as indicated by the manufacturer  polymer and a thiol-functionalized crosslinker (CD-Link) which comprises a matrix-metalloproteinase cleavable peptide allowing embedded cells to cleave the crosslinker and migrate within the gel. Mixing the two components leads to the formation of stable thioether bonds and formation of the gel. Gelation time varies with pH and can be adjusted by mixing two ten-fold concentrated buffers (10× CBpH 7.2 and 10× CBpH 5.5). The lower the pH, the slower the gel formation. Peptides containing cell adhesion motifs such as RGD can be covalently attached to a fraction of the thiol-reactive groups of the thiol-reactive polymer, either prior to or after crosslinking to support adhesion of cells. Since crosslinking with CD-Link causes the pre-gel solution mixture to start to solidify in less than 3 min at room temperature, it was added to the reagent mixture just before the gel filling process was started. The prepared gel complex was placed on ice and the MFC was placed on a cold surface for gel filling to maximize the gelation time as much as possible.2 at 37 °C in T75 cell culture flasks, and passaged by dissociation with 0.05% trypsin/EDTA every two to three days at a confluency of 70–90%. Cell suspension for the experiments was prepared by centrifugation of dissociated cells at 130× g for 7 min at RT.HT29 Human colon adenocarcinoma cells were cultured and maintained at 5% CO6 cells/mL. HT29 cells were seeded at ≈1600 cells per abluminal channel as each channel was filled with ca. 4 μL gel.A cell strainer with a pore size of 40 μm was employed to extract single HT29 cells in suspension, which is crucial to generate similarly sized tumor spheroids in the MFC. Each 30 μL volume of hydrogel composition comprised 6 μL cell suspension at a density of 2 × 10A fresh microfluidic chip was UV sterilized for 30 min. The gel was infused into the channels using a previously sterilized syringe pump set-up as described above. After gel filling, the MFC was placed in an upright position under the sterile hood and incubated for ca. 60–90 min to allow for gelation.TM Red (Invitrogen) prior to seeding and cultivation in the microchip to enable real-time visualization of the interaction of tumor spheroids  with the MDCK cell barrier. The dye was dissolved in 20 μL high-quality DMSO to prepare a stock solution at RT; 3 μL of this stock solution was diluted in 500 μL PBS to prepare the CellTrackerTM Red working solution. The dye is well retained in living cells through several generations.MDCK cells stably expressing a Vimentin specific chromobody  . After cell seeding, the reservoirs were covered with Parafilm and the chip was placed in an upright position in the incubator for approximately 2 h to facilitate the adhesion of cells to the gel surface. After incubation, fresh medium was added to the reservoirs and the perfusion setup was switched on. The medium was changed once every two days. The formation of a cellular barrier across the gel interface and the interaction with tumor spheroids was monitored regularly using a fluorescence microscope (Nikon Eclipse Ti).Each channel was filled with 10 μL of MDCK cell suspension   adhered 2  enabled remodeling by 3D tumor spheroids and their outgrowth into the perfusion channel.The incubator-compatible perfusion system enables gravitation-driven unidirectional flow vs. bidirectional flow as found in other models . It inteHowever, the need for certain improvements also became apparent: in a future design, fluid connectors could be implemented in a standard industry format and spacing on the chip. Silicone tubing used with peristaltic pumps may cause unwanted absorption of compounds ,42 and iThe feasibility and versatility of cell culture in the MFC in general was demonstrated by cultivation of the cellular barriers of MDCK, and also in the co-culture with HT29 tumor spheroids in the adjacent gel phase.TEER measured using the integrated electrodes generally was significantly lower than that seen in earlier reports ,54,55. WThe inhibition of cell growth in HT29 tumor spheroids under perfusion at the beginning of the culture may be due to the unintended dilution of factors secreted by the cells in case of an excess liquid to cell ratio and highlights the issue of finding and establishing the proper liquid to cell ratio (LoC) in microfluidic in vitro models . As relaIn summary, we demonstrated a parallelized microfluidic device and a comprehensive workflow to study and analyze co-cultures of cellular barriers and 3D tumor spheroids under continuous unidirectional flow.