Synedra, fungal microparasite Zygophlyctis, and co-growing bacteria), and even ≥17-fold in field-sampled populations . Additional data obtained using the Synedra–Zygophlyctis model system reveals that fungal infections reduce the formation of aggregates. Moreover, carbon respiration is 2-fold higher and settling velocities are 11–48% lower for similar-sized fungal-infected vs. non-infected aggregates. Our data imply that parasites can effectively control the fate of phytoplankton-derived organic matter on a single-cell to single-aggregate scale, potentially enhancing remineralization and reducing sedimentation in freshwater and coastal systems.Phytoplankton forms the base of aquatic food webs and element cycling in diverse aquatic systems. The fate of phytoplankton-derived organic matter, however, often remains unresolved as it is controlled by complex, interlinked remineralization and sedimentation processes. We here investigate a rarely considered control mechanism on sinking organic matter fluxes: fungal parasites infecting phytoplankton. We demonstrate that bacterial colonization is promoted 3.5-fold on fungal-infected phytoplankton cells in comparison to non-infected cells in a cultured model pathosystem (diatom  Fungal parasites are found to effectively control the fate of phytoplankton-derived organic matter, potentially enhancing remineralization and reducing sedimentation in freshwater and coastal systems. Most of this decaying organic matter is remineralized within the euphotic zone, but a critical fraction gets exported to deeper layers as sinking particulate material in diverse systems, including freshwater4 and coastal environments6. Particulate organic matter is thereby transported through the water column towards sediments—a mechanism that drives the bentho–pelagic coupling, i.e., the exchange of energy, mass, and nutrients between pelagic and benthic habitats. Sinking organic matter thus sustains life below the euphotic zone7, while, on the other hand, it can also lead to expanding oxygen-deficient waters worldwide in areas where oxygen consumption through organic matter remineralization exceeds the available oxygen supply10. The fate of phytoplankton-derived organic matter is, therefore, key to aquatic food webs and biogeochemical cycles.Phytoplankton grows fast, renewing its biomass once per week and leaving behind several gigatons of decaying organic matter in the water column of the aquatic biosphere11. In this regard, sinking aggregates are primary vehicles of (in-)organic matter to depth12. The formation, remineralization, and sedimentation of aggregates are commonly explained by physical and biological processes, the latter being intimately linked to zooplankton grazing, bacterial degradation, and viral lysis16. Recent data, however, suggests that we are still missing some crucial links in the network of biological control mechanisms on vertical organic matter fluxes17. Aquatic fungi, for instance, are hardly considered in this context although they are abundant and active across various aquatic systems19, reaching from high-mountain lakes20 over coastal areas21 to the deep sea22. For instance, members of the fungal division Chytridiomycota, referred to as chytrids, can thrive as microparasites on phytoplankton cells. These chytrids are traditionally well documented in lakes23, especially during bloom events when host abundances are high25. More recently, they have also gained increasing attention in coastal systems after being observed via microscopy, for instance, in the highly productive upwelling region off Chile26, in the Arctic Ocean28, and during harmful algae blooms in the Mediterranean Sea29. Using high-throughput sequencing methods, Chytridiomycota have also been detected in other marine regions34, and DNA-based abundances of Chytridiomycota have been correlated to phytoplankton blooms or chlorophyll in coastal regions38. Chytrids thus occur frequently in freshwater and coastal regions where a strong benthic–pelagic coupling is commonly assumed39, whereas in open ocean regions, their distribution is less evident with rare observations so far40.After blooming, phytoplankton cells together with fecal pellets and other detritus can coagulate as aggregates—referred to as lake or marine snow—which sink rapidly with velocities of up to several hundred meters per day24 or even 80–100% of their specific phytoplankton host population43, including diatoms, dinoflagellates, and cyanobacteria. They thereby alter the dynamics of phytoplankton populations44 and are presumed to impact organic matter remineralization and sedimentation45. In a natural lake, chytrid-infected phytoplankton has been shown to mainly decompose in surface waters, whereas non-infected cells settled to depth41. It has further been estimated that 20–25% of the photosynthetically derived carbon from the infected host population is channeled to parasitic chytrids 46 and further to zooplankton (through the mycoloop)47. Chytrids have therefore been proposed to be an integral part of aquatic food webs and element cycling48. Yet, their impact on the fate of phytoplankton biomass in terms of aggregate formation and vertical organic matter fluxes remains unknown.Parasitic chytrids infect up to 1–44%50 and often mediate high proportions of the particle export52, are highly susceptible to fungal infections in freshwater and coastal systems44. We thus used one of the few available diatom–parasite model pathosystems (freshwater diatom Synedra and chytrid Zygophlyctis)53 to investigate the impact of fungal infections on the formation and characteristics of sinking diatom aggregates and associated organic matter fluxes. We hereby considered cell aggregation, mass density, settling velocity, sticky polymers, bacterial colonization, and respiration—parameters that are all intimately linked to organic matter remineralization and sedimentation processes. Moreover, we complemented these culture-based investigations with analyses of bacterial abundances and infection prevalence in aggregates that were formed from field-sampled phytoplankton communities. Our results demonstrate that epidemics of fungal microparasites need to be appreciated as an episodically impactful control mechanism on organic matter fluxes in the aquatic biosphere.Diatoms, which form fast-sinking aggregatesSynedra sp. and chytrid Zygophlyctis planktonica pathosystem are indicated as model system and data from the field-sampled populations as natural systems.Data obtained from the cultured diatom Synedra and co-growing bacteria. The designated infected treatment additionally included the parasitic chytrid Zygophlyctis, which developed Synedra-associated sporangia and free-living zoospores, both being part of the life cycle of this fungal microparasite53. One set of both treatments (each in triplicates) was grown and sub-sampled over a growth period of nine days, to monitor the culture development (referred to as culturing). An additional set was grown for six days and thereafter transferred into rotating cylinders, to analyze the formation and characteristics of aggregates (referred to as rotating cylinders). Symbols, terms, and abbreviations used in the following are summarized in Supplementary Box We incubated non-infected and chytrid-infected diatom co-cultures—hereafter non-infected and fungal-infected treatment, respectively. Both culture treatments included the freshwater pennate diatom Synedra cells  on maturely-infected cells. Post-infected Synedra cells displayed remains of chitinous cell walls as a sign of previous infections after zoospore discharge. Decaying cells displayed no chlorophyll autofluorescence and also no signs of previous infections, and thus, they had most likely not undergone any fungal infection46.For cell enumeration, we distinguished non-infected, early-infected, maturely-infected, post-infected, and decaying lls Fig. . Non-infSynedra abundances increased from approximately 0.8 × 104 to 3 × 104 cells mL−1  reached 47% on day 6 (mostly maturely-infected cells) and remained at the same percentage until day 9 , with similar chlorophyll a contents in both treatments during day 0–2  but significantly lower chlorophyll a contents in the infected vs. non-infected cultures during day 4–9 .During the 9-day growth period, total L−1 Fig. , with si56. Photometrically-analyzed TEP concentrations increased until day 6 and decreased thereafter in both treatments . TEP concentrations were significantly higher (P < 0.001) on day 2, but significantly lower on day 9  in the infected treatment in comparison to the non-infected treatment. On the other sampling days, no significant differences were detected between both treatments (P > 0.22). Similar to TEP concentrations, CSP concentrations increased until day 6 and decreased thereafter . Microscopy-derived numbers of TEP and CSP were highly variable, ranging from 119 to 3672 TEP mL−1  and from 966 to 21,196 CSP mL−1  in both culture treatments .Transparent Exopolymer Particles (TEP) and Coomassie Blue Stainable Particles (CSP) were determined microscopically and spectrophotometricallySynedra cells. On day 9, the abundance of diatom-associated bacteria was lowest in conjunction with non-infected Synedra cells (6.5 ± 4.2 bacteria diatom−1) and increased with increasing infection stage, from early-infected to maturely-infected, post-infected, and decaying Synedra cells . Consequently, diatom-associated bacteria were in total 3-times more abundant in the infected culture  and day 6 (P = 0.0001) in the infected culture. On day 9, however, the abundances of free-living bacteria were similar in both treatments  free-living bacteria as well as diatom-associated bacteria based on their association with (ii) non-infected, (iii) early-infected, (iv) maturely-infected, (v) post-infected, and (vi) decaying ure Fig. . Abundanvs. non-infected Synedra treatments  were 38 ± 14-times less abundant  and comprised 46 ± 24-times less cumulative aggregate volume  in the fungal-infected Synedra treatment. Thereafter, also large aggregates (>1.3–5.6 mm) became abundant, and both size classes (d = 0.4–1.3 mm and >1.3–5.6 mm) were 6 ± 4-times less abundant (range: 2–14-times) and comprised 9 ± 7-times less cumulative aggregate volume  during fungal infections. Aggregates within the smallest size bin (d = 0.35–0.46 mm) were most abundant  accounted for most of the cumulative aggregate volume 57, was significantly higher for non-infected aggregates than for infected aggregates  was significantly different between non-infected and infected aggregates . This indication, however, was not confirmed by similar porosities of non-infected and infected aggregates , but carbon-specific respiration rates were on average 2-fold higher for fungal-infected aggregates as compared to non-infected aggregates 63. Due to the slower settling velocities and higher respiration rates, infected aggregates were estimated to respire 2.4‒3.7-times more carbon per meter settled than non-infected aggregates  and diatoms . Chytrid infections were highest on Planktothrix, Synedra, and Fragilaria cells, and thus, we inspected these taxa in more detail 24. Bacterial abundances were ≥17-times higher on infected cells than on non-infected cells .We sampled a natural phytoplankton community in a temperate lake (Lake Stechlin), which comprised mostly filamentous cyanobacteria , but the stickiness, i.e., the probability that two cells coagulate upon encounter, was presumably reduced during fungal infections. The stickiness of cells and particles can be increased by the presence of exopolymers like TEP and CSP, which are broadly classified as acidic polysaccharides and alkaline amino acids, respectively89. TEP concentrations showed significantly different dynamics over time in our non-infected vs. infected Synedra culture  fewer freely-suspended polymers, reducing the overall aggregation, but (2) more adhesive polymers at the cell surface of infected Synedra cells, enhancing their cell-to-cell stickiness. Indeed, the observed faster growth of bacterial populations in the infected treatments  than the field-sampled cells (Lake Stechlin), and their morphologies were different . The different aggregation patterns between cultured and field-sampled Synedra may thus have resulted from genera-specific differences (involving diatom-specific bacteria interactions) or even strain-specific differences, as known from viral infections on phytoplankton95. Moreover, in culture, post-infected Synedra cells showed a higher aggregation potential than early-infected and maturely-infected cells  as compared to siliceous frustules (1.82 g cm−3)97. The lower mass density of infected Synedra cells and aggregates in comparison to non-infected cells and aggregates may further be explained by more cell-associated polymers (such as TEP), fewer cells per aggregate, or thinner silica frustules as bacteria are known to dissolve silica98. Following Stokes’ law (Eq. d = 1–5 mm) sank 11–48% slower than non-infected aggregates. And secondly, the herein-measured lower mass densities (measured in a density gradient) and lower excess densities (Eq. vs. non-infected aggregates would both result in ca. 30% slower settling velocities of infected aggregates (following Stokes’ law), similar to the size~settling velocity regression. We thus conclude that host-associated sporangia increased the cell size of diatom cells but reduced their mass per volume (g cm−3), slowing down their settling. Reduced aggregate formation and hence, smaller aggregates during fungal epidemics may have an even stronger impact on sinking velocities. For example, maximum aggregate diameters were 1 mm in the non-infected treatment but only 0.5 mm in the infected treatment after 6 h of aggregate formation. Such a 2-fold decrease in aggregate diameter led to 3-fold slower sinking velocities  would have lost 37% and non-infected aggregates only 12%  of their initial carbon content after settling 50 m, indicating a substantial reduction in export efficiencies due to fungal infections.Aggregates that comprised 71% fungal-infected cells respired twice as much of their carbon content per day as compared to non-infected aggregates with similar size and POC content Fig. . We presies Fig. , we roug44, including productive upwelling regions26, commercial mass cultures101, and areas impacted by harmful algal blooms29, their epidemics thus have the potential to diminish the strength and efficiency of vertical organic matter fluxes in natural and engineered aquatic environments.Taken together, our incubations revealed that fungal infections decreased the chlorophyll content, cell mass density, cell aggregation, aggregate size, and settling velocity, but increased bacterial colonization and carbon respiration on a single-cell to single-aggregate scale Fig. . As a reSynedra sp. Ehrenberg, 1830  and the parasitic chytrid Zygophlyctis planktonicum (strain SVdW-SYN-CHY1), including host-associated sporangia  and zoospores  isolated from lakes in Northern Germany53. Bacteria were co-isolated with the diatom and chytrid and maintained in co-culture in a nutrient-replete medium for several months, which likely selected for copiotrophic taxa46. Chytrid infections on the diatom host were initiated by free-swimming zoospores, which attached to a host cell, encysted, and developed into epibiotic sporangia while penetrating and digesting the host’s interior through a rhizoidal system53. Within the (zoo-)sporangium, the next generation of zoospores was formed and finally released through the rupture of the sporangium. One infection cycle took 1–2 days. Each infection was lethal and prohibited further reproduction of the host.The model pathosystem comprised the pennate diatom host −1m−2 during the 16-h light phase. Synedra was grown as 12 × 650 mL in 1 L Erlenmeyer flasks until reaching ca. 7500 cells mL−1. Half of those flasks (6 × 650 mL) were thereafter inoculated with a Synedra–Zygophlyctis co-culture , including mature fungal sporangia, which were projected to discharge new zoospores within the next hours (infected treatment). The resulting infection prevalence was 9% in the infected treatment on day 0. The other 6 × 650 mL flasks remained without Zygophlyctis (non-infected treatment). To monitor the culture development over time, 6 × 650 mL (three per treatment) were grown and sub-sampled for nine days (see culture characteristics). The remaining 6 × 650 mL (three per treatment) were grown for six days and subsequently transferred into rotating cylinders to follow the formation of aggregates (see formation and characterization of sinking aggregates). Erlenmeyer flasks were gently shaken by hand once per day for cell resuspension and water mixing.Batch cultures were grown in CHU-10 medium  after 0, 2, 4, 6, and 9 days. CHU-10 medium served as a blank for all analyses.a and nutrients analyses, 3 mL were transferred into 15 mL Falcon tubes and centrifuged at 3000 rpm for 20 min at 4 °C. The supernatant was gently removed and the remaining pellet was extracted in 90% acetone, frozen at −20 °C, and analyzed after one month using a fluorometer . Concentrations of chlorophyll a were calculated and corrected for phaeopigments, measured after acidification103. Calibration was done with a chlorophyll a standard . Concentrations of nitrate/nitrite and soluble reactive phosphorous were determined from 0.45  µm-filtered water by flow injection analysis (FIA) and spectrometric detection . Concentrations of dissolved organic carbon (DOC) were analyzed from GF/75-filtered water on a TOC-V CPH with a nondispersive infrared sensor  using combustion catalytic oxidation.For chlorophyll Synedra cells were counted in Utermöhl plankton chambers  under an inverted epifluorescence microscope . The chitinous cell walls of sporangia were stained with Calcofluor White 104. Synedra cells were differentiated as (i) non-infected, (ii) early-infected, (iii) maturely-infected, (iv) post-infected, and (v) decaying cells  and Wheat Germ Agglutinin 104. Bacteria were counted under a fluorescence microscope (Leitz Leica DMRB) at x1000 magnification. For each group and replicate, 20 Synedra cells (for attached bacteria) or 20 counting grids (for free-living bacteria) were examined, to reach representative mean values (standard error ≤5%).For bacterial abundance analyses, 0.5–1 mL were preserved with paraformaldehyde , filtered onto polycarbonate filters , and stored at −20 °C. Prior analyses, filters were stained with 4′,6-diamidino-2-phenylindole  onto PC filters . TEP filters were stained with Alcian Blue  for 5 s, and CSP filters with Coomassie Brilliant Blue G  for 30 s. After staining, filters were rinsed with MilliQ to remove excess dye and stored at −20 °C. Sterile-filtered water (0.2 µm) from the cultures was used as blank. For spectrophotometric analyses, AB was extracted in 80% H2SO4 and CBB-G in 3% SDS in 50% isopropyl alcohol, as specified in Supplementary Methods −1) and CSP concentrations relative to bovine serum albumin .Transparent Exopolymer Particles (TEP) and Coomassie Blue Stainable Particles (CSP) were determined microscopically and spectrophotometrically105. Images were corrected for uneven light intensities in the background and split into separate RGB channels. The blue channel was subtracted from the red channel, to remove chlorophyll-related areas and outlines of diatoms cells. The obtained 8-bit greyscale images were reversed in color and a global threshold was applied. Automated particle analyses in ImageJ provided the number of particles per filter area  and the cross-sectional area A of each particle . Particles with ECD < 3 µm were not included. Since the automatic TEP and CSP recognition was not perfect, the processed images were compared visually with the original images, and falsely outlined TEP or CSP were manually removed from the data set. Cell outlines and the infection stages were poorly visible on our images, and thus, we removed all cell-associated TEP and CSP outlines to not introduce any bias. Only freely-suspended TEP and CSP were therefore included in the microscopy analyses, whereas the spectrophotometric analyses included freely-suspended and cell-associated TEP and CSP.For microscopic analyses, the stained filters were placed onto CytoClear slides and inspected under a microscope . For each filter, 40 images were taken randomly along two transects across the entire filter area. Care was taken to use the same microscope and camera settings during the entire image acquisition. Image analyses were done in ImageJ 1.51p−3). The viscous layers were prepared with mixtures of Ludox TM colloidal silica , sucrose, and distilled water49. After 12 h and an additional centrifugation run , cells had settled into the density layer that was equivalent to the mass density ρs-cells of their particulate material when hydrated in liquid 57. The intact density layers (visible to the naked eye) were sub-sampled into 2 mL tubes and stored at 4 °C. Half of each sample was analyzed for its mass density ρs-cells (g cm−3) using a density meter (Anton Paar DMA 38). The other half was used to enumerate non-infected and infected diatom cells (after CFW staining) under an inverted fluorescence microscope with combined bright-field and UV excitation .To analyze the mass density of diatom cells (on day 6), 10 mL were transferred into 15 mL Falcon tubes and centrifuged . The supernatant was gently removed, and cells were re-suspended in 1 mL CHU-10 medium and transferred onto a density gradient, consisting of six viscous layers with increasing density from top to bottom (1.18–1.38 g cmN = 3 flasks) in the infected treatment. Thereafter, the cultures were diluted with CHU-10 medium to approx. 5000 cells mL−1 (resembling cell abundances during bloom scenarios87) and transferred into rotating cylinders  to mimic the aggregation of cells due to their stickiness and differential settling behavior  but their physical properties such as density, porosity, and composition are comparable to natural aggregates108. Initial diatom abundances were similar in both treatments . Cylinders were rotated at 1–2 rpm at 17 °C in darkness on a roller table. The aggregate formation was recorded over time using a Mini Deep-focus Particle Imager . The MDPI is a ‘shadowgraph imager’ using a near-infrared LED light source  behind a pinhole and a set of identical plano-convex collimator lenses to create parallel light beams between the LED light pod and the camera pod . The sampling volume of two image sequences covered 2 × 0.35 L, equal to 30% of the entire cylinder volume.Triplicates of non-infected and fungal-infected cultures were grown for six days until the infection prevalence had reached 59 ± 3% . We counted the number of aggregates per size class, reported as aggregate number concentration N(d) (# L−1). To describe the number concentration as a function of size, the aggregate size spectrum n(d) (# L−1 mm−1) was calculated as∆d (mm) is the width of each size class 110. The volume spectrum nVd was calculated by normalizing the volume distribution to the aggregate sizeVagg (mm3) is the average volume of single aggregates in each size class111. Vagg and d  were calculated from A derived from the image analyses and assuming spherical geometry. After stopping the rotation, individual aggregates were picked with a wide-bore glass pipette for various analyses. To ensure that aggregate sizes were similar for non-infected and infected aggregates, aggregates were sampled after 30 h from the non-infected treatment and after 45 h from the non-infected treatment . Aggregates were counted, measured, and binned into size classes, in which the upper diameter was 1.3-times the lower size class. The resulting eleven size classes ranged from 0.4 to 5.6 mm 57. We measured the settling velocity U of aggregates in a settling column . The column was filled with the same water as the rotating cylinders and kept at the same temperature (17 °C). It was double-walled, with a 2 cm interspace filled with water and a sealed top, except for a 2 cm inlet to introduce the aggregates. This setup minimized convective currents in the column during settling experiments112. Each aggregate was allowed to freely settle from the wide-bore pipette into the water. The settling was timed along 15 cm using a stopwatch. Excess densities ∆ρ of aggregates were calculated using Stokes’ law, which applies to ballasted phytoplankton aggregates113ν is the kinematic viscosity (1.085 × 10−2 cm2 s−1 at 17 °C) and g the gravitational acceleration (981 cm s−2). The drag coefficient CD and Reynolds number Re derived fromSet I was used to determining the size, porosity, excess density, settling velocity, and fractal dimension. Aggregates were imaged directly after sampling using a compact camera . The cross-sectional area D3. D3 was derived from the slope of d and U after log–log transformation  respiration rates by assuming a respiratory quotient of 1.2 mol O2 to 1 mol CO2115 and by normalization to the aggregate-specific POC contents. Shifts in oxygen concentrations due to, e.g., temperature changes, were tested in control vials with 0.2 µm-filtered water but without aggregates. The observed shift in oxygen concentration was significantly lower in the control vials than in the vials with aggregates . After incubations, aggregates were filtered individually onto pre-combusted GF/F filters  and stored at −20 °C for later POC analyses. After storage, filters were fumed over HCl and dried at 50 °C overnight. POC was analyzed after combustion using a carbon analyzer . Carbon respiration rates are given in the units d−1 and % m−1  analyses. Single aggregates were transferred into 5.9 mL gas-tight Exetainer® vials with 0.2 µm-filtered water from the rotating cylinders. Oxygen concentrations were measured with a Clark-type oxygen microsensor  immediately after aggregates were added and after 24 h-incubations at 17 °C in darkness. During incubations, the Exetainer rotated to keep the aggregates freely suspended and to minimize diffusion-limited gas exchange at the aggregate surfacem−1 Fig. , which dfit Fig.  and meanfit Fig. .Synedra populations was analyzed in Lugol-preserved samples (x400 magnification), whereas abundances of Synedra-associated bacteria were analyzed in PFA-preserved samples (x1000 magnification). Aggregates were disaggregated via gentle shaking in 2 mL microcentrifuge tubes prior to filtration and/or counting, to be able to inspect individual Synedra cells under the microscope. The number of aggregate-associated bacteria (per aggregate) was calculated by multiplying the number of associated bacteria per Synedra-cell type with the abundance of each Synedra-cell type per aggregate. We assumed 20,000 Synedra cells per aggregate since we counted on average 22,843 ± 34,443 Synedra cells per aggregate with d = 2.1 ± 0.8 mm (N = 18).Set III and IV served to determine the infection prevalence and bacterial abundance. Single aggregates and 1 mL of the ambient water were sampled and inspected under the microscope, as described above (see section Culture characteristics). In short, the infection prevalence in ρs-agg. Single aggregates were gently transferred on top of a density gradient. The further procedure resembled the protocol used for ρs-cell, i.e., after centrifugation and settling, the layer containing the aggregate was sampled and analyzed for its mass density .Set IV was used to derive the mass density of single aggregates d = 2–4 mm) were formed. Individual aggregates were sampled with a wide-bore glass pipette, and sub-volumes of the ambient water were sampled with a plastic syringe. Ten replicates (10x aggregates and 10 × 2 mL ambient water) were sampled from each cylinder and split in half. One half was preserved for determining the infection prevalence on single phytoplankton taxa, and the other half for counting cell-associated bacteria. Sample preservation and microscopy analyses were done as described above (see section Culture characteristics).A plankton community was sampled from Lake Stechlin (Northern Germany) during the spring bloom in April 2018. Cells were concentrated from 0–10 m using a hand-held plankton net , resuspended in 10 L of in situ surface water, and transferred into three rotating cylinders. Cylinders rotated at 7.5 °C  for 12 h in darkness until macroscopic aggregates . Normal distribution was tested using the Shapiro-test and data variance with the F-test. Statistical differences between multiple groups were determined with the Kruskal–Wallis test . Statistically significant differences between time series data were calculated based on generalized linear mixed models . Data were log-transformed if the assumption of homogeneity of variance was rejected . Pair-wise comparisons in time series data were run with emmeans (v. 1.7.2 in R). Statistical tests and plotting were done in R 3.3.0116 and Origin2021. Statistical tests were run two-sided. The significance level was 0.05. P values at very highly significant levels are given as <0.001 or <0.0001. Uncertainties (±sd) that derived from combined uncertainties of single variables were calculated following the laws of error propagation. The number of replicates is indicated for each mean value in the results section.Pairwise comparisons between mean values were calculated with the Mann–Whitney test for non-normally distributed data, the Welsh-test for normally distributed data with non-equal variance, and the Further information on research design is available in the Peer Review FileSupplementary MaterialsDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


EM) population and restricting the expression of tumor suppressors in a preclinical model of spontaneous colitis-associated colorectal cancer (CAC). We show that IFN-γ expression is significantly increased both in the T cells and the colonic mucosal epithelia of mice with a T cell-restricted deletion of the TGF-β intermediate, SMAD4 (Smad4TKO). The increase of IFN-γ expression correlates with the onset of spontaneous CAC in Smad4TKO mice by 6 months of age. This phenotype is greatly ameliorated by the introduction of a germline deletion of IFN-γ in Smad4TKO mice . DKO mice had a significantly reduced incidence and progression of CAC, and a decrease in the number of mucosal CD4+ TEM cells, when compared to those of Smad4TKO mice. Similarly, the colon epithelia of DKO mice exhibited a non-oncogenic signature with a decrease in the expression of iNOS and p-STAT1, and a restoration of the tumor suppressor gene, 15-hydroxyprostaglandin dehydrogenase (15-PGDH). In vitro, treatment of human colon cancer cells with IFN-γ decreased the expression of 15-PGDH. Our data suggest that Smad4-deficient T cells promote CAC through mechanisms that include an IFN-γ-dependent suppression of the tumor suppressor 15-PGDH.Immune cells and the cytokines they produce are important mediators of the transition from colitis to colon cancer, but the mechanisms mediating this disease progression are poorly understood. Interferon gamma (IFN-γ) is known to contribute to the pathogenesis of colitis through immune modulatory mechanisms, and through direct effects on endothelial and epithelial homeostasis. Here we explore whether IFN-γ influences tumor progression by expanding the effector memory T cells (T Colorectal cancer (CRC) is the third most commonly occurring cancer and is the second most common cause of cancer-related deaths in the world , 2. Glob+ T lymphocytes  develop chronic inflammation in the intestinal mucosa, a phenotype associated with spontaneous CAC (+ effector memory T (TEM) cells (Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with important functions for the maintenance of immune homeostasis and is one of the key molecules regulating epithelial cell biology and immunity in the gut . Inadequeous CAC , and theM) cells . These rTKO model (The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is an enzyme responsible for the degradation of PGE2 into an inactive metabolite . 15-PGDHKO model . The synKO model . While TKO model , 33, infKO model .In vitro IFN-γ treatment suppresses tumor suppressor 15-PGDH in cultured colon cancer cells. Smad4TKO mice exhibit mucosal epithelial hyperplasia that is accompanied by increased inflammation in colonic mucosa and a significant reduction in the expression of 15-PGDH in colon epithelial cells. Germline deletion of IFN-γ in Smad4TKO mice  significantly reduces the incidence and progression of CAC via a decrease in the population of CD4+ TEM cells and a restoration of 15-PGDH expression in colon epithelia, when compared to Smad4TKO mice. These data demonstrate the direct link between alterations of TGF-β signaling in T cells and malignant transformation in epithelial cells in the gastrointestinal tract, through an IFN-γ-dependent alteration in the expression of the tumor suppressor 15-PGDH.Here we report that in the absence of TGF-β signaling in T cells, the production of proinflammatory cytokines is significantly increased and IFN-γ expression is down-regulated through a Smad4-dependent mechanism. Anti-phospho-Stat1 (Tyr701) (58D6), anti-phospho-Stat3 (Thyr705) (D3A7), anti-phospho-iκB (14D4), anti-iNOS (D6B6S), and anti-PD-L1 (D5V3B) were purchased from Cell Signaling. Anti-CD3, anti-CD28, anti-CD25, anti-CD44, anti-CD62L, anti-IFN-γ, IL-6, and anti-TNF-α were purchased from BD Biosciences. Anti-Foxp3 antibody was purchased from eBioscience.SMAD4 gene  in mice has been described previously (KO) was purchased from The Jackson Laboratory. To generate mice deficient for both IFN-γ germ line and for Smad4 in the T cell lineage only, IFN-γKO mice were crossed with Smad4TKO mice. The resulting F1 heterozygotes were then bred to generate all genotypes, including Smad4TKO/IFN-γKO (DKO) mice. Mice were housed in a specific pathogen-free facility. All animal experiments were performed in accordance with institutional guidelines and with approval of the Institutional Animal Care and Use Committee at Case Western Reserve University.T cell-restricted deletion of the eviously , 32. TheThe colon was excised from the ileocecal junction to the anal verge, flushed with phosphate-buffered saline (Invitrogen) and opened longitudinally. Gross examination was performed to evaluate tumor size and number and to measure colon length and colon weight. The colon length to colon weight ratio was measured to assess thickening of the intestinal mucosa. The incidence , the mean tumor size ± standard deviation, and the mean number of tumors/mouse ± standard deviation were calculated for each group. Tumor size was determined by image analysis using imaging software (ImageJ). Images were taken with a scale bar and lengths were measured in pixels and correlated to the known distance in scale bars. Colon tumors as well as colonic tissues were processed for histopathological evaluation and further biochemical analyses.Serum Nitric oxide (NO) levels were measured by photometric analysis by using a nitrite/nitrate assay kit , according to manufacturer’s instructions.-ΔΔCt, in which ΔΔCt equals ΔCt of the experimental sample  minus ΔCt of the control sample (WT mouse sample).Colon mucosa was obtained from scrapings of full-length colon and total RNA was isolated using Trizol reagent (Invitrogen). For reverse transcription-PCR (RT-PCR), cDNA was synthesized using a High Capacity cDNA synthesis kit (Applied Biosystems). Quantitative RT-PCR was performed using BioRad CFX96 Real-Time System C1000 Thermal Cycler. The expression of target genes was normalized to expression of housekeeping gene β-actin. The relative gene level was expressed as 22. FET cells were seeded in 12-well plates at 1 x 105 per well as triplicates and transiently transfected with 0.2 mg of SBE promoter vector or 2.5 kb 15-PGDH –PGL promoter vector and 20 ng of CMV-renilla using LipofectAMINE Plus as transfection agent according to the manufacturer’s instructions (Invitrogen). Approximately 24 hrs past transfection, cells were treated with IFN-γ (20 ng/ml) or IL-6 (20 ng/ml) for 24 hrs in medium. Luciferase activity was measured using Promega Dual Luciferase Assay Kit  and a ML3000 Microtiter Plate Luminometer. Data shown represent the mean of three independent experiments. For IFN-γ promoter luciferase activity, pCMV5 plasmid or pCMV5-Smad4 plasmid were transfected one day before transfection of 3.6 Kb IFN-γ promoter in 293T, Jurkat or primary murine T cells and 24 hrs after IFN-γ-promoter transfection, luciferase activity was measured. For T cell activation, T cells were plated on the plate coated with anti-CD3/CD28.The FET human colon carcinoma cells were cultured in MEM (Invitrogen) with 10% fetal bovine serum (Germini) and glutamine (2 mg/ml) at 37 °C in 5% COFor Western blot, colon mucosa was obtained from scrapings of full-length colon and lysed by incubation in lysis buffer  on ice for 30 min. 20 μg aliquots of proteins were separated by electrophoresis in 10% SDS/PAGE minigels and transferred to nitrocellulose membrane (Invitrogen). Following blocking, membranes were incubated in buffer containing the primary antibody, followed by washing and incubation for 1hr at room temperature with horseradish peroxidase-conjugated secondary antibodies. Immunostaining was visualized by ECL.For hematoxylin and eosin staining (H&E), excised colons were washed with PBS and fixed in 10% formalin. Samples were embedded in paraffin wax, sectioned, stained with H&E, and examined by light microscopy. For immunohistochemistry (IHC), slides were deparaffinized and rehydrated and heat-induced epitope retrieval was performed prior to blocking with Peroxidazed 1  and Rodent Block M . Slides were incubated with primary antibodies, CD3 and PD-L1 for one hour at room temperature. Antibodies were detected using Rabbit-on-Rodent HRP polymer  and visualized using Betazoid DAB chromogen kit . The percentage of CD3 positive cells were assessed on six randomly selected field using digital eyepiece.LP immune cells were isolated using Mouse Lamina Propria Dissociation Kit (Miltenyi Biotec). Colon sections were washed clean, cut into small pieces, and incubated with 5 mM EDTA, 1 mM DTT (MilliporeSigma), and 5% FBS in HBSS buffer (Thermo Fisher Scientific) for 20 minutes. Then tissue was collected into gentle MACS C tube (Miltenyi Biotec) and dissociated to single cells using gentleMACS Dissociator (Miltenyi Biotec).Cell suspensions were prepared from spleens or colon lamina propria by filtering through nylon mesh (40-µm diameter). Erythrocytes were lysed using ACK lysis buffer (BioWhittaker) and cells were washed twice in RPMI 1640 supplemented with 10% heat-inactivated FBS, 50 µM 2-ME, penicillin, and streptomycin (Invitrogen). Viable cells were counted using trypan blue exclusion on a hemocytometer. All Antibodies used in FACS analyses were purchased from BD Pharmingen. Pan T cells were purified from spleen and lymph node using a Pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions (purity greater than 95%).in vitro by plate-bound anti-CD3 and anti-CD28 antibodies in 24-well plates in the absence or presence of TGF-β for 72 hrs. The cells were harvested, washed with PBS, stained with anti-CD4 and CD8 antibodies, and stained for intracellular Foxp3 using a Foxp3 staining Kit (eBioscience) according to the manufacturer’s instructions.For intracellular staining for cytokines, lymphocytes from spleen and colon lamina propria were activated with plate-bound anti-CD3 and anti-CD28 antibodies for 48 hrs and re-stimulated with PMA and ionomycin for the last 5 hrs in the presence of Golgi stop solution prior to washing and staining with antibodies for CD4 and CD8. Intracellular staining for IFN-γ, IL-6 and TNF-α was performed using an intracellular staining kit (BD Biosciences) according to the manufacturer’s instructions. For inducible regulatory T cell assay, splenocytes from each genotype were activated p-value less than or equal to 0.05, with *p<0.05, **p<0.01, ***p<0.001.Data are expressed as means ± SE. Statistical significance was determined by 1-way ANOVA with Tukey–Kramer Multiple Comparisons Test. The Fisher’s Exact Probability test was used for comparison of the incidence of lesions between the two groups. Statistical significance was accepted to be a co/co;Lck-cre, Smad4TKO) leads to spontaneous mucosal inflammation, and epithelial cancers throughout the gastrointestinal tract. Smad4TKO mice develop CAC, with the majority of tumors arising after 6 months of age  cells (CD44HighCD62LLow) was significantly increased (78.74%) compared to wild type mice (35.29%)  cells in the colon of Smad4TKO was decreased (17.74%) compared with that of WT (27.71%) while there were no significant changes in the splenic Treg population  through a T cell lineage-restricted deletion of the Smad4 gene in mice . In this35.29%) , 35, 36,.29% , 35(35.29%) . In a T EM cells and the malignant transformation of colonic epithelium in Smad4TKO mice, we examined the expression levels of tumor suppressors in the colon of Smad4TKO mice. The expression level of the tumor suppressor 15-PGDH was significantly reduced in the colons of Smad4TKO mice, implying that soluble factors from the pathogenic TEM may be responsible  onto a genetic background with a germ line IFN-γ deletion (IFN-γKO) to generate a ‘double knockout’  model. DKO mice harboring the T cell-restricted deletion of the tumor suppressor SMAD4 and a germ line deletion of proinflammatory cytokine IFN-γ ameliorated CAC and inflammatory infiltration of the mucosa as early as 8 months of age, at which point the mortality rate of DKO mice began to decrease compared with the Smad4TKO mice. The survival rate of DKO was 75% at the age of 10 months, compared to 38% for Smad4TKO mice  in DKO was significantly smaller than that (2.3 mm) in Smad4TKO mice. Furthermore, tumor multiplicity was less than 1 tumor/mouse in DKO mice, whereas more than 4 tumor/mouse were found in Smad4TKO mice. DKO mice showed a similar phenotype as WT or IFN-γKO, with a delayed disease presentation (12 months) as compared with the Smad4TKO (8 months). These data clearly demonstrate that germ line IFN-γ deletion decelerates CAC development and tumorigenesis in the Smad4TKO mice. Histological analysis of intestinal sections from Smad4TKO mice revealed disrupted villus architecture with regions of epithelial atypia, as well as adenomas and invasive carcinomas as compared to the colon histology of the WT, IFN-γKO and DKO mice  cells in cancer is complex, and is often dysregulated within the TME  were validated by introducing the germ line deletion of the IFN-γ gene into Smad4TKO mice . Utilizing the DKO mouse model, we discovered that the CAC phenotype in Smad4TKO mice is linked to gain of IFN-γ expression in lymphocytes, with significant skewing of the mucosal CD4+ T cell repertoire toward an activated, effector memory phenotype. However, other types of cells, such as CD8 T cells, NK cells, and NKT cells cannot be ruled out as a potential important source of IFN-γ expression in this model system. Invariably, the colonic epithelium of Smad4TKO mice exhibited an inflammation-driven oncogenic signature that includes a significant suppression in the expression of 15-PGDH and an elevation in the expression of iNOS, p-Stat1 and p-Stat3.In this study we have demonstrated a role for IFN-γ production by Smad4-deficient pathogenic T cells in the pathogenesis of inflammation-related CRC in mice. Our data support the finding that IFN-γ abundance accelerates gastrointestinal epithelial malignancy by promoting epithelial cell transformation through suppressed production of the tumor suppressor 15-PGDH in the colonic epithelial cells as well as enhanced production of pro-inflammatory mediators by tissue resident CD4In vitro IFN-γ treatment of cultured colon carcinoma cells suppressed 15-PGDH expression  and concomitant suppression of 15-PGDH expression in epithelial cells. Importantly, strategies designed to modulate the expression and activity of soluble factors in both the epithelial and stromal compartment in the TME may serve to concomitantly support the maintenance of mucosal epithelial homeostasis and suppress the expansion of pathogenic, tissue-resident effector T cells producing inflammatory cytokines, thereby forming a unique and effective approach to cancer immunotherapy.To our knowledge, this is the first report describing dual, tandem mechanisms for tumor promotion by IFN-γ, that include the expansion of pathogenic effector CD4The original contributions presented in the study are included in the article/The animal study was reviewed and approved by The Institutional Animal Care and Use Committee at Case Western Reserve University.SC, JL, and B-GK designed the studies and developed the methodology; SC and B-GK performed experiments; SC, AH, JL, and B-GK interpreted the data; SC and B-GK wrote the initial draft of the manuscript; SC, AH, JL, and B-GK edited the manuscript and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Institutes of Health grants (R01CA168586 and 1R03CA259901-01A1).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.All claims expressed in this article are solely SFthose of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pathologies of the blood–brain barrier (BBB) have been linked to a multitude of central nervous system (CNS) disorders whose pathology is poorly understood. Cortical spreading depression (CSD) has long been postulated to be involved in the underlying mechanisms of these disease states, yet a complete understanding remains elusive. This study seeks to utilize an in vitro model of the blood–brain barrier (BBB) with brain endothelial cell (b.End3) murine endothelioma cells to investigate the role of CSD in BBB pathology by characterizing effects of the release of major pronociceptive substances into the extracellular space of the CNS. The application of trans-endothelial electrical resistance (TEER) screening, transcellular uptake, and immunoreactive methods were used in concert with global proteome and phospho-proteomic approaches to assess the effect of modeled CSD events on the modeled BBB in vitro. The findings demonstrate relocalization and functional alteration to proteins associated with the actin cytoskeleton and endothelial tight junctions. Additionally, unique pathologic mechanisms induced by individual substances released during CSD were found to have unique phosphorylation signatures in phospho-proteome analysis, identifying Zona Occludins 1 (ZO-1) as a possible pathologic “checkpoint” of the BBB. By utilizing these phosphorylation signatures, possible novel diagnostic methods may be developed for CSD and warrants further investigation. Changes in the extracellular milieu of the brain are reported during neurological disorders, including low pH and high extracellular potassium ,2. For eThe BBB is comprised of cerebral capillary endothelial cells, locked together by tight junction  and adherens junction  proteins to create a dynamic, highly selective permeable barrier between the blood and CNS ,25,26. D2 standard flasks  with Dulbecco’s modified eagle media (DMEM) . DMEM was supplemented with 2 μM L-glutamine , 10% fetal bovine serum (FBS) , and 1% penicillin-streptomycin ). C8-D1A murine astrocytes  were cultured in 75 mm2 standard flasks with DMEM supplemented with 10% (FBS)  and 1% penicillin-streptomycin . Both cell lines were split upon reaching 80% confluence to prevent overgrowth. Cell culture flasks were then incubated in a 37 °C humidified incubator with 5% CO2:95% air atmospheric conditions. b.End3 murine immortalized endothelial cells  were cultured under sterile conditions in 75 mm2O, 148.19 mM NaCl, 3 mM KCl, 1.85 mM CaCl2, 1.71 mM MgCl2 1.80 mM NaHPO4, 229.20 µM NaH2PO4) in ACM at equivolume to 60 mM KCl was used as a control (vehicle); (2) ACM buffered to a pH of 6.8 to model release of H+ ions into the extracellular space at the CSD wave front, prepared by titrating ACM down to a pH of 6.8 ± 0.05 with 12 M HCl ; (3) glutamate  dissolved in ACM at concentrations of 10 μM, 30 μM, and 100 uM; and (4) 60 mM KCl  dissolved in ACM, serving as a positive control. A total of 60 mM KCl treatments at relevant physiological levels were utilized as it is a typical condition used to evoke K+-ion-triggered spreading depolarization in live brain slices [b.End3 endothelial cells utilized in these studies were cultured in astrocyte conditioned media (ACM) for at least 24 h prior to any type of treatment, collection, or fixing. The usage of ACM for endothelial co-culture was integral to the functional culture of an endothelial barrier by supplying the critical growth factors and modeling the in vivo critical role of astrocytes in the maintenance of proper endothelial barrier function. ACM was produced in house by culturing fresh DMEM (Gibco 11995-065) cell culture medium supplemented with FBS  and penicillin 100 UI/mL-streptomycin 100 μg/mL  for 24 h with a confluent growth of C8-D1A mouse astrocytes. This media was aliquoted and frozen at −20 °C for use when needed. Culturing endothelial cells in ACM allows for the formation of a functional cell monolayer and tight junctions in vitro. To model a CSD event, cells were treated with a 5 min pulse of one of the following: (1) artificial cerebrospinal fluid (aCSF)  uptake assays [2:95% air atmospheric conditions. Upon the formation of a luminal monolayer on the Transwell insert, co-cultures were then used for downstream analyses.In vitro modeling of the BBB was performed on a Transwell monoculture system, utilized for TEER, e assays . b.End3 n = 3.The TEER technique utilizes measured changes in electrical resistance between two chambers filled with an aqueous solution and separated by a cultured cell barrier, with increased electrical resistance indicative of increased barrier integrity, as the free flow of ions between the chambers is prevented by the cellular barrier, manifesting as an increased electrical resistivity due to loss of electrical conductance between the chambers. a. b.End3TM fluorescent secondary antibodies  in 70% ethanol for one hour and air-dried them for 30 min under a UV lamp in a fume hood. Dried coverslips were placed in a 12-well plate and treated with a 20% collagen solution for two hours. Collagen solution was then removed by vacuum suction, and b.End3 cells were aliquoted in 80 μL volumes of DMEM media and incubated at 37 °C until the formation of a monolayer, after which a 24 h ACM incubation was initiated. Cells were then washed in 1× PBS and treated with one of the following preparations: (1) vehicle (aCSF in ACM), (2) ACM buffered to pH = 6.8, or (3) 60 mM KCl in ACM. After treatment cells were washed with 1× phosphatate buffered saline , then fixed with a 1% paraformaldehyde solution, permeabilized with 0.2% Triton X-100  in 1× PBS for 10 min at room temperature, and blocked in a 10% bovine serum albumin (BSA) solution  with 0.1% Triton X100 for 1 h. Primary antibodies  prepared2:95% air to allow the formation of a cell monolayer, upon which abluminal media was replaced with ACM and another 24 h incubation initiated. After incubation treatment, the pulses consisted of the following: (1) vehicle (aCSF in ACM), (2) ACM buffered to a pH = 6.8, and (3) 60 mM KCl in ACM. Pulse treatments were aliquoted in triplicate into a new 24-well plate, with inserts transferred from the original culture plate to the treatment plate for 5 min of treatment. A 30 min time course was initiated, and abluminal media was collected at 5 and 30 min timepoints in new collection plates. Scintillation vials for radiolabel quantification were prepared with Optiphase Supermax cocktail  to act as a suspension agent. Radiolabeled 14C-sucrose  was prepared by suspending 100 µL stock 14C-sucrose in 10 mL of DMEM, and 50 µL of this preparation was assayed for the working range of a radioactive emission of 50,000 counts per minute (CPM) ± 15,000. Once prepared, 14C-sucrose suspension was added to the luminal side of each insert, which was then immediately subjected to an abluminal treatment pulse, then transferred to a collection plate containing abluminal ACM. Abluminal media from the 5- and 30 min collection plates was then collected and aliquoted into a 5 mL scintillation vial , placed on a scintillation counter, and allowed to run overnight to capture CPM values. Samples were run in triplicate for each condition for n = 4. All radioactive material was disposed of according to University of Arizona regulations (RAM Protocol #698).b.End3 cells were seeded on the luminal side of Transwell inserts pretreated with 20% calf collagen and incubated at 37 °C with 5% CO2 for 72 h until the formation of an endothelial cell monolayer on the insert. Upon the formation of the monolayer, 1000 µg/mL of 4 or 70 kDa fluorescein isothiocyanate-dextran1  solution was prepared in DMEM. Once prepared, the FITC preparation was added to the luminal side of Transwell inserts and pulsed abluminally for 5 min with the following treatments: (1) vehicle (aCSF in ACM), (2) ACM buffered to pH = 6.8, and (3) 60 mM KCl in ACM. All abluminal treatment media was then removed and replaced with fresh ACM, and a 180 min time course for the experiment was initiated. Aliquots of 10 µL were then removed from abluminal wells of each insert at timepoints of 10, 20, 30, 60, 120, and 180 min, and diluted into 90 μL DMEM to allow for a working volume and placed into a black clear-bottom microplate for fluorescence reading. Fluorescence readings were obtained with a ClarioStar plate reader (BMG Labtech) at an excitation wavelength of λ = 483 nm and emission wavelength of λ = 530 nm. The experiment was repeated 4 times in triplicate for an overall n = 4.b.End3 cells were seeded onto the luminal side of Transwell inserts pretreated with 20% calf collagen. Inserts were cocultured with abluminal ACM and incubated at 37 °C in 5%:95% air CO2:95% air and grown to confluence. Growth media was then removed and replaced with ACM, and cells were incubated for an additional 24 h. Cells were removed, washed with 1× PBS, and treated with a 5 min pulse of either (1) vehicle (aCSF in ACM), (2) ACM buffered to a pH = 6.8, and (3) 60 mM KCl in ACM. Treatments were removed and cells were then washed in 1× PBS, and 200 μL of cell lysis buffer  containing 1% by volume of protease and phosphatase inhibitor cocktail  was added to each well and harvested via cell scraping. Lysed cell material was then added to a new 1.7 mL microfuge tube and centrifuged for 10 min at 13,000× g at 4 °C. Supernatant was then transferred to a new 1.7 mL microfuge tube for protein quantification.b.End3 cells were seeded on 6-well culture plates and incubated at 37 °C with 5% COProtein quantification was performed using a Pierce BCA protein assay kit . Quantification standards were prepared from a 2 mg/mL Pierce BSA Standard  serially diluted for the generation of quantification curve. Albumin standards and cell lysate samples were pipetted into a microplate in duplicate, and working reagent was added to each sample, covered, and incubated at 37 °C for 30 min. Subsequently, an incubation plate was assayed for optical density (OD) on a BMG ClarioStar plate reader at λ = 562 nm. OD values for BSA standards were used to generate a quantification curve, which was used to calculate the total protein concentration in each sample. Samples were then diluted to a concentration of 1 μg/μL in lysis buffer with 5× Lane Marker Reducing Sample Buffer  and Dithiothreitol (DTT) . Samples were then frozen at −80 °C.® 20 Detergent  for 5 min, then blocked in 5% milk in 1× tris-buffered saline  for 30 min. Primary antibodies were then added to the membrane (prepared in 5% BSA in 1× TBS), and the membrane was incubated at 4 °C for 48 h on a rocker. Following incubation, primary antibodies  were loaded into a criterion gel electrophoresis cell, then filled with sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-Page) running buffer . Wells were then loaded with 30 μg of previously prepared cell lysate samples in triplicate by treatment group. An electrophoresis cell was run at 150 V for 10 min, then 190 V for 40 min. Following electrophoresis, gels were removed from their plastic cassettes and placed into a membrane transfer apparatus with a nitrocellulose membrane  and filled with SDS transfer buffer . The voltage was set at 20 V and transfer was run for 120 min. Following the transfer, apparatus was disassembled and the nitrocellulose membrane was removed and washed 3 times in 1× tris-buffered saline with 0.1% Tweentibodies  were remtibodies  at room 2O), and (3) one 3 min wash with 500 µL no-salt buffer (10 mM tris-HCl pH = 7.5 in H2O). Pellets were then eluted into 60 µL 2× Laemmli dye  with 10% DTT, and heated to 95 °C for 10 min, cooled, and loaded onto a 10% SDS-PAGE gel for electrophoresis, then placed into a transfer apparatus with a nitrocellulose membrane for the transfer of proteins to the membrane. Following the transfer, the membrane was blocked in 5% milk, and incubated with anti CL-5 antibody at 1:500  vehicle (aCSF in ACM), (2) ACM buffered to pH = 6.8, and (3) 60 mM KCl in ACM. Treatments were removed, and cells were washed in 1× PBS at pH 8.0 and chilled to 4 °C. NHS-SS (Succinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate) biotin linking reagent buffer from a Thermo Cell Surface Isolation Kit  was then added to cells and incubated at 4 °C for 25 min. A fresh aliquot of biotin buffer was added, and cells were incubated at 4 °C for another 25 min. Following incubation, the biotin buffer was removed, and cells were washed with pH = 8.0 buffered 1× PBS. Cell lysis buffer was prepared in house with protease and phosphatase inhibitors added at a ratio 1:100 and added to cells. Cell lysate was then scraped off the culture plates and transferred to a 1.7 mL microfuge tube and incubated on ice for 1 h. Subsequently, incubation tubes were centrifuged at 14,000 RPM for 10 min at 4 °C and supernatant was pooled by treatment group into a new 1.7 mL centrifuge tube. Neutravidin beads from the Cell Surface Isolation Kit were then equilibrated in lysis buffer and added to the supernatant previously collected. Tubes containing supernatant and beads were then sealed with parafilm and incubated overnight at 4 °C on a rocker to allow for mixing. The following day, each tube with was re-aliquoted into a new 1.7 mL microfuge tube and centrifuged at 14,000 RPM for 5 s to pellet Neutravidin beads. After removing and freezing supernatant, Neutravidin beads were washed following the following protocol at 4 °C: (1) three 3 min washes with 500 μL of lysis buffer, (2) two 3 min washes with a high-salt buffer . The search variables used were as follows: 10 ppm mass tolerance for precursor ion masses, and 0.5 Da for product ion masses, trypsin digestion, maxima of two missed tryptic cleavages, variable modifications of phosphorylation of threonine, tyrosine, and serine, and oxidation of methionine. Scaffold software  was used to cross-correlate Mascot search results with X! Tandem software. Significance value was set at p ≥ 0.05. Ion intensity-based label-free quantification was performed using Progenesis QI for proteomics software . Raw files were imported and converted into two-dimensional maps with y axis defined as time and x axis defined as m/z, which was then followed by the selection of a reference run for alignment. The aligned runs were then used to create an aggregate data set containing all peak information from all samples, after which the data pool was narrowed down to only +2, +3, and +4 charged ions for further analyses. The top 8 most intense precursors of a given feature were grouped into a peak list of fragment ion spectra and exported in a Mascot generic file (.mgf) and searched against the Mus musculus SwissProt_2018_01 database utilizing Mascot software. The following search variables were used: 10 ppm mass tolerance for precursor ion masses and 0.5 Da for product ion masses, trypsin digestion, maxima of two missed tryptic cleavages, variable modifications of oxidation of methionine, and phosphorylation of serine, tyrosine, and threonine, 13C = 1. The data were collected into a Mascot .xml file and imported into Progenesis allowing for the assignment of peptides and proteins. Peptides with a Mascot ion score < 25 were not used for further analyses. Non-conflicting peptides and precursor ion abundance values were normalized using a reference run to perform protein quantification. A heat map of principal component analyses (PCAs) and unbiased hierarchal clustering analyses were performed in Perseus [b.End3 murine endothelial cells were treated with either a 24 h hormone treatment or pulsed with one of the CSD constituent substances, as previously described. b.End3 cells were then lysed post treatment and loaded into SDS-PAGE for separation by electrophoresis. A total of 200 μg of harvested cell lysate supernatant was separated on a 10% SDS-PAGE gel and stained for total protein presence with Bio-Safe Coomassie G-250 Stain. Lanes from the gel were separated and cut into six slices, which then underwent trypsin digestion, and resulting peptides were purified by C18 desalting performed as described by Kruse et al. . High-pe Perseus ,34.n = 4) underwent tryptic digestion and enrichment of phosphopeptides with sequential enrichment from metal oxide affinity chromatography, as per the manufacturer’s instruction (Thermo Scientific). A Thermo Orbitrap Fusion Lumos Tribrid Mass Spectrometer fitted with an EASY Spray source was used to perform HPLC-ESI-MS/MS in positive-ion mode, according to the manufacturer’s protocol. NanoLC was performed using a Thermo Scientific UltiMate 3000 RSLCnano System with an EASY Spray C18 liquid chromatography column ; loading phase for 15 min at 0.300 mL/min; mobile phase, linear gradient of 1% to 34% buffer B in 119 min at 0.220 mL/min, followed by a step to 95% buffer B over 4 min at 0.220 mL/min, hold 5 min at 0.250 mL/min, and then a step to 1% buffer B over 5 min at 0.250 mL/min and a final hold for 10 min ; buffer A 5 0.1% formic acid/H2O; and buffer B 5 0.1% formic acid in 80% acetonitrile. All solvents utilized were liquid chromatography mass spectrometry grade. Xcalibur software  was used to acquire Spectra, and Progenesis QI  was used to perform ion-free intensity-based label-free quantification. .raw files were imported and converted into two-dimensional maps with y axis defined as time and x axis defined as m/z, which was then followed by a selection of a reference run for alignment. The aligned runs were then used to create an aggregate set containing all peak information from all samples, after which the data pool was narrowed down to only +2, +3, and +4 charged ions for further analyses, which were then grouped by treatment. A peak list of fragment ion spectra was generated and exported in Mascot generic file (.mgf) and searched against the Mus musculus SwissProt_2018_01 database, utilizing Mascot software with the following search variables: 10 ppm mass tolerance for precursor ion masses and 0.5 Da for product ion masses, trypsin digestion, maxima of two missed tryptic cleavages, variable modifications of oxidation of methionine, and phosphorylation of serine, tyrosine, and threonine, 13C = 1. Protein or peptide assignment was performed by importing the resultant Mascot .xml file into Progenesis, while peptides with a <25 Mascot ion score were not used further. Precursor ion abundance values for peptide ions were normalized to all proteins. Differences were assessed as significant if a difference between vehicle and treatment groups was p < 0.05 assessed with one-way analysis of variance (ANOVA). Consensus phosphorylation sequences were determined using iceLogo [b.End3 cells were cultured and treated as previously described. A total of 5 mg of protein lysate per sample  policy (NOT-OD-15-102), so that differences of 20% were detected with 80% power at a significance level of 0.05. Post-experimental data analyses were performed in GraphPad Prizm 7.0 (GraphPad Software). Unless otherwise noted, data were analyzed with either a two-way paired or unpaired Pronociceptive substances released at the wavefront of CSD events were screened to qualitatively assess the significant induction of a paracellular leak in the BEB. Substances found significant were then assayed for their functional impact on BEB with quantitative transport assays to assess the magnitude of the BEB breach and functional outcomes on BEB.p = 0.0010) and acidified media (** p = 0.0011), (p = 0.2941) and 100 μM ATP (** p = 0.0058) demonstrated no significance, and a gradual drop in TEER became significant 20 min following treatment, respectively. The rapid reduction in TEER values following KCl and acidified pH delayed the effect of ATP, and no effect from glutamate suggests a fast-acting mechanism regulating BEB functionality.The integrity of the BEB was assayed via TEER a,b to as0.0011), b, mainta14C-sucrose, 4 and 70 kDa FITC uptake assays were performed over a 30 and 180 min time course, respectively, to assess the functional alterations to the BEB as well as quantifying the magnitude of the BEB breach. Both 4 and 70 kDa FITC uptakes demonstrated no significant differences between treatments  and acidified pH treatment (**** p < 0.0001)  c, which  0.0001) d. This oOur previous in vivo studies indicated that the total detection of expressed TJ proteins CL-5 and OCC was unchanged in isolated microvessels following CSD induction . To detep = 0.0351) reducing and acidic pH (* p = 0.521) increasing CL-5 CTCF. These treatments had no significant effect on both ZO-1 and VE-CAD fluorescence. These observations suggest the rapid onset of apical protein relocalization in vitro.Alterations to the localization of the proteins CL-5, ZO-1, and VE-CAD were assayed to observe changes at the TJ (CL-5), AJ (VE-CAD), and intracellular compartment (ZO-1) of the endothelial cells following KCl and acidified pH insult a. Confocp = 0.0415), indicative of potential homeostatic stress response following KCl insult.The structural integrity of the endothelial cell TJ complex is dependent on intracellular actin cytoskeletal linkage to transmembrane TJ proteins, such as CL-5 and OCC via ZO-1, 2, and 3. Cells undergoing structural insult can be identified by the increased detection of filamentous actin (F-actin) vs. normal globular (G-actin) actin. FITC-conjugated phalloidin was used to stain f-actin filaments following KCl and acidified pH treatment a,b. FollCL-5 has recently been validated as a clinical biomarker for several CNS disorders; therefore, focus was placed on this protein to further characterize its utility as a tool for investigating alterations to TJ integrity under experimental conditions, and to further characterize the potential of clinical diagnostic use. Biotynylation cell surface protein isolation assays were utilized to quantify the changes in surface localization of CL-5 following KCl and acidified pH insult. p = 0.0231) was shown to increase the surface detection of CL-5 vs. vehicle  to the global proteome. p < 0.0544). Acidified pH downregulated 68 proteins and upregulated 160 proteins (p = 0.2286) and ZO-1 upregulated following KCl exposure (* p = 0.0207) (p = 0.0034) while downregulating Caza2 (p = 0.0006) and Caz2b (p = 0.0508). KCl exposure significantly downregulated Caza2 (p < 0.0001). Taken together, these data indicate that the loss of BEB integrity is due to a synergistic convergence of unrelated individual deleterious processes induced by KCl and acidified pH exposure to TJ proteins CL-5, ZO-1, and actin maintenance proteins AFAP1, Caza2, and Cap2b. Global proteome analysis of b.End3 cells post treatment detected a total of 7113 proteins, 6279 of which were identified as being statistically significant  D. Degrad 0.0207) D. Actin p-values to visualize the functional effect on pathways and processes related to TJ and cytoskeletal homeostasis  and Kyoto Encyclopedia of Genes and Genomes (KEGG) bioinformatics databases to assess the functional impact of protein expression changes detected in our analysis following KCl and acidified pH exposure. Separated by treatment group, significant proteins (α < 0.05) were queried through the following three GO bioinformatic databases: biological processes (BPs), cell compartment (CC), and molecular function (MF). Additionally, the KEGG pathway database was screened, and positive hits were scored by fold enrichment (FE), with the top ten from each database listed, separated by treatment group, and graphed by FE score against log transformed -Log10 eostasis a,d. Dataeostasis D. These Phosphorylation of CL-5 is associated with increased permeability of the BBB, but it is unknown if CSD-like conditions induce these post-translational modifications (PTMs). Following treatment with vehicle, KCl, and acidified pH, cells were quantified for total phosphorylation enrichment and amino acid residue enrichment of phosphorylation sites by individual protein. A total of 25,546 total phosphorylation adducts were detected, of which 11,371 were deemed significant by 3-way ANOVA (α = 0.05). When analyzed by treatment group, 237 proteins experienced phosphorylation following vehicle treatment vs. KCl and acidic pH A. A totap-values  as opposed to Evans Blue and the exclusive use of 70 kDa FITC. These differences in methodology may have contributed to the opposing findings contained herein. The variable detection of F-actin stress fibers following KCl and acidified pH insult was confirmed in the global proteomic analysis. The expression of actin-associated proteins AFAP1 and Caza2 and Cap2b were significantly altered following acidic pH exposure, and KCl heavily downregulated Caza2. Mirroring the treatment-dependent functional changes observed at the TJ, differential molecular mechanisms may underlie these changes. The findings from total proteome analyses following modeled CSD insult indicate a potential major homeostatic disruption to several key structural proteins in endothelial cells, evidenced by a significant alteration to the enrichment of actin-associated proteins AFAP1, Caza2, and Capzb, in addition to CL-5 and ZO-1 changes. The discrete manner by which each specific CSD constituent plays a role in these observations warrants further investigation into a potential PTM mechanism.These proteomic data seem to contrast with our previous report : the preData from this study demonstrate that acidic pH dynamically regulates more proteins and phosphorylation PTMs than KCl in murine brain endothelial cells centered on the downregulation of actin reorganization and capping, while upregulating responses to exocyst organization and negative regulation of membrane tubulation. Phosphorylation of proteins related to adheren junction’s reorganization and localization was also downregulated by acidified pH. KCl globally facilitated microtubule formation by downregulating depolymerization at the signaling level by upregulating actin formation pathways. This treatment-dependent effect was further validated by the absence of overlap in total protein enrichment between treatments, suggesting changes in brain pH and K+ concentration increase BEB permeability via distinct mechanisms. Phosho-proteomic analysis indicated PTMs as a likely mechanism for divergent treatment response, as phosphorylation is a reversable and highly dynamic mechanism of protein regulation. ZO-1, a major linking protein between the cytoskeleton and TJ was significantly enriched in phosphorylated residues specific to each treatment resulting in similar functional outcomes. This functional convergence on ZO-1 from both treatments may also indicate a central regulatory role for ZO-1 in response to CSD-like insult; specifically when considering the critical function, it performs maintaining structural homeostasis. Therefore, similar functional outcomes due to divergent KCl and acidified pH phosphorylation sites on ZO-1 demonstrate a potential mechanism driving protein relocalization and the paracellular leak of the BEB.Low brain pH and high extracellular K+ during neurological disorders promote BEB/BBB paracellular leak by inducing TJ and actin cytoskeletal reorganization and functional alteration through independent mechanisms. The elucidation of these molecular mechanisms may be utilized as a “diagnostic fingerprint” specific to a unique pathology potentially useful as a biomarker.


Restriction on hospital visits for COVID‐19 infection control continues to have a significant negative impact on patients and their families. For a patient receiving palliative care, this social isolation may deteriorate their mental health. In such situations, home care could be a viable solution to this problem. In the COVID‐19 pandemic, providing end‐of‐life care at home may allow patients to spend more time with their families, improve communication between them and their health professionals and reduce the possibility of hospital‐acquired infections. In an attempt to limit the spread of COVID‐19 infection, hospitals and nursing homes have imposed visiting restrictions, which prevent patients from spending precious time with their loved ones in their final days of life. This separation might further worsen the mental health of both vulnerable patients and their family members.Home care could be a preferable solution to this problem. Home care is a form of care in which patients stay at home with the help of their families, nurses, care workers, doctors and other health care professionals. This form of care is actively practiced in some countries, including Japan, where reimbursement for home care is available, including for patients with cancer and palliative care needs.2A man in his late 80s living in Tokyo with his wife and two daughters experienced sudden abdominal pain in July 2020 and went to seek help in a nearby tertiary hospital. That same day, he underwent an emergency laparotomy and appendicectomy after being diagnosed with generalized peritonitis associated with the perforation of an appendiceal tumor. Once the diagnostic tests were conducted, the patient was confirmed to have Stage IV appendiceal cancer. Postoperative contrast‐enhanced computed tomography (CT) scan did not show any abnormal findings such as local recurrence, lymphadenopathy, or distant metastasis associated with cancer. However, histopathological tests revealed scattered lymphatic invasion in the sub‐serous layer. Accordingly, the patient was offered palliative care instead of invasive procedures such as surgery.Family members were informed about the severity of the disease highlighting the need for end‐of‐life care. However, due to COVID‐19, the hospital imposed certain restrictions, which limited the number of visitors it can allow per patient. Only one family member was allowed to visit an inpatient, and only with approval from the hospital or physician. Accordingly, the family started seeking updates on the patient's condition through phone calls with the medical staff. However, family members were unable to comprehend all information clearly, leading to misunderstandings and even conflicts between healthcare staff and family members. For example, there was an incident where the family members thought that the intravenous drip was administered through the peripheral intravenous catheter, but later they found it was administered through the peripherally inserted central venous catheter. So, family members were having difficulty adjusting to such incidents. They were particularly concerned when hospital staff asked them not to call too often and were told that medical staff would contact them directly if anything happened to the patient.acute respiratory distress syndrome (ARDS) secondary to heart failure. The patient received well‐organized, proficient home‐based health care, and spent the final days of his life with his family, friends, and other loved ones.The patient's family was worried about home care initially because the hospital told them it would be difficult and expensive. However, considering the patient's desire to spend his final period at home with his family, along with the restriction on hospitals visits due to the pandemic, the patient and his family chose to receive professional home care services in November 2020. After his discharge, he started receiving home care where home care nurses visited twice a day and the home doctor visited once every 2 weeks. Moreover, two daughters assisted his wife as family caregivers, and professional caregivers provided clinical care through a publicly funded nursing care insurance scheme. The patient was able to spend ample time with their loved ones, which was not possible in the hospital. On weekends, his grandchildren and great‐grandchildren also visited him, and the whole family was able to spend quality time with the terminally ill patient. Moreover, communication between the healthcare professionals and the family was also enhanced. Nurses communicated on a daily basis, and the doctor in charge made house calls as needed. In April 2021, the patient passed away peacefully after experiencing 3This case illustrates the importance of home‐based end‐of‐life care for cancer patients, particularly during crises such as the COVID‐19 pandemic, as it allows patients to spend quality time with their families along with optimal care if managed effectively. Typically, end‐of‐life care is provided around the globe in hospice or hospital, aiming to provide holistic, personalized care to patients and to support families and caregivers.In our case, the terminally ill patient was able to enjoy the comfort of his own home, cherish his lifetime memories, and spend quality time with his loved ones. Besides the psychosocial implications, we believe that there are several other reasons to choose home‐based end‐of‐life care during a COVID‐19 pandemic. First, this would prevent communication problems between healthcare professionals and family members. Restrictions imposed due to COVID‐19 have reportedly reduced the quality of communication between hospitalized patients and their family members, resulting in severe distress that may affect the quality of death and bereavement.However, home care also has its drawbacks. Previous studies have reported challenges related to delivering urgent care when necessary4The findings of this case study highlight that in crises situations such as COVID‐19, home care could be considered as a viable option for terminally ill cancer patients.Sakamoto R, Bhandari D, Ozaki A, Yoshida M, and Tanimoto T wrote the manuscript. All authors conceptualized and designed the study and contributed to making critical revisions for improving the intellectual content of the manuscript.AO reports personal fees from Medical Network Systems, MNES Inc. TT reports personal fees from Medical Network Systems, MNES Inc. and Bionics co. Itd. Other authors declare no competing interests.Written informed consent was obtained from the patient's child.The patient is deceased and we obtained a consent form from the patient's child.


Endothelial cell density variation was 5.51%, 3.06% and 2.82% in groups A, B and C, respectively (p = 0.19). Total Endothelial Cell Loss (ECL) was 4.37%, 5.32% and 7.84% in groups A, B and C, respectively (p = 0.39). Endothelial cell morphology was comparable in all three groups. Conclusions: In the DMEK RAPID Mini, low temperatures (<2 °C) may affect the quality of pre-loaded grafts, inducing a higher ECL after 72 h of preservation, although no significant differences among groups could be proved. Our data would suggest maintaining grafts loaded in the DMEK RAPID Mini at temperatures between 2–8 °C for appropriate preservation.The aim of the study was to assess different temperature ranges for the preservation of pre-loaded Descemet Membrane Endothelial Keratoplasty (DMEK) grafts in the DMEK RAPID Mini device. Methods: Three groups of 15 DMEK grafts (five per group) were pre-loaded in the DMEK RAPID Mini and preserved in Optisol-GS for 72 h at different temperatures: group A at >8 °C, group B between 2–8 °C and group C at <2 °C. After stripping and preservation, the viability of the endothelium, cell loss and morphology were assessed through light microscopy following trypan blue and alizarin red staining. Results: Overall mortality was 4.07%, 3.97% and 7.66%, in groups A, B and C, respectively, with percentages of uncovered areas of 0.31%, 1.36% and 0.20% (all  Eye banks are playing a crucial role in the development of more standardized and surgeon-friendly techniques for preparing, loading, transporting and delivering grafts for Descemet Membrane Endothelial Keratoplasty (DMEK) ,2,3,4. IIn the eye bank, the DM can be loaded inside the injector either with the corneal endothelium folded outwards (ENDO-OUT configuration) or inwards (ENDO-IN configuration). Once separated completely from the posterior stroma, the DM naturally adopts a scrolled, ENDO-OUT configuration, an effect likely due to the different degrees of elasticity of the anterior and posterior surfaces of the DM . InsteadEye banks have been collaborating with surgeons to provide pre-trephined and pre-loaded membranes preserved either with ENDO-IN or ENDO-OUT configurations. To improve such preparations, efforts are being made to minimize Endothelial Cell Loss (ECL) associated with preparation ,3,12,13,While no dedicated device is so far available for membranes folded inwards, the DMEK RAPID Mini system  has recently received CE marking for the transportation of pre-loaded, ENDO-OUT grafts . This syThe aim of this study was, therefore, to evaluate the effects of preservation at different low-temperature ranges on corneal endothelial cells viability, simulating the transport of pre-loaded DMEK grafts in the DMEK RAPID Mini device.Donor corneal tissues unsuitable for transplantation due to poor endothelial cell counts, procured by Fondazione Banca degli Occhi del Veneto Onlus  were used for the research purposes and the validation studies described in this manuscript, according to the realms of the law 91/99 and after an informed consent form was signed by the donor’s next of kin.n = 15) retrieved from the morgue and preserved in Cold X Medium (homemade medium) at 4 °C for up to 3 days. The membranes were prepared following a standard stripping protocol as previously reported [In this study, we used fifteen corneas ;Group A: >8 °C storage (-n = 5);Group B: 2–8 °C storage in a “transport simulation” condition (-n = 5).Group C: <2 °C storage  and stored at different temperatures, as described below:Transport simulation was performed by leaving the pre-loaded tissues on a laboratory shaker  placed inside a temperature-monitored refrigerator for the entire duration of the experiment. All the membranes were preserved for 72 h.2, a mortality rate lower than 2.5% and percentages of uncovered areas lower than 1% at T0.The tissues were analyzed after the stripping step (T0). The membranes were stained with trypan blue dye and evaluated by means of light microscopy  using a hypotonic analysis solution (HAS) to highlight the intercellular borders and to assess Endothelial Cell Density (ECD), cell mortality rate, acellular areas . To be included in this study, tissues had to have an ECD > 1700 cells/mmAfter 72 h of preservation (T72), the membranes were carefully extracted from the injector, gently placed and unfolded on a glass slide, stained with trypan blue dye and immersed in hypotonic analysis solution to perform the second light microscopy evaluation.In general, trypan blue positive cells appear within the corneal endothelium as a result of different possible discomfort events or situations, such as poor initial quality of the tissue, long post-mortem time, long storage time, stressful storage conditions, iatrogenic trauma, etc. All these conditions lead to changes in the cell membrane permeability, allowing the dye to stain the cellular nuclei. These cells are considered dead and ready to detach from the Descemet membrane. Uncovered (or acellular) areas are a natural consequence of cellular deaths or are caused by severe mechanical traumas leading to cell detachment .2 squares of a 10 × 10 mm reticule inserted in the eyepiece of an inverted microscope were counted manually at 100× magnification. The number of trypan blue positive cells allowed us to determine the percentage of cell death and uncovered areas (2) and 8.25 mm (53.43 mm2). For the confirmation of uncovered areas, Alizarin red  was topically applied, and the tissues were incubated for 5 min at RT away from bright light. The tissues were then re-evaluated under the light microscope.To estimate the ECD and the extent of damaged areas, the cells in five 1 mmed areas  in relatThe overall mortality was obtained by calculating the difference between the mortality detected at T72 (post-preservation) and the mortality found at T0 (post-stripping). The overall uncovered areas were calculated using the same method. By doing so, the damage that occurred during the stripping phase could be excluded from the final data evaluation, which will be only referred to in the conservation phase. Total ECL was obtained by combining these two values .Descriptive data were summarized using the mean (standard deviation) and percentages where appropriate.The normal distribution of outcomes was checked by means of Shapiro–Wilk tests and visual inspection of Q-Q plots.One-way ANOVA was used to compare the effect of the 3 temperature groups on ECD, mortality rate and uncovered areas at T0 and at T72, as well as ECD variation, overall mortality, overall uncovered area and total ECL between T0 and T72. Homogeneity of variance and covariance was confirmed by means of Levene’s test and Box’s M-test, respectively.t-tests with Bonferroni correction were used for comparisons between groups in case of a significant ANOVA result.Post hoc pairwise p-value of 0.05 was considered statistically significant. All analyses were conducted using R software version 4.2.2 .A Membranes with similar biological features were used for all the groups .2, the mortality rate was 0.26 ± 0.31% and the percentage of uncovered areas was of 0.16 ± 0.27%. In Group B, the average ECD was 2110 ± 260 cells/mm2, the mortality rate was 1.02 ± 0.21% and the percentage of uncovered areas was of 0.07 ± 0.16%. In Group C, the average ECD was 2060 ± 134 cells/mm2, the mortality rate was 0.54 ± 0.47% and the percentage of uncovered areas was of 0.14 ± 0.28% (After stripping (T0), in Group A the mean ± SD ECD was 2160 ± 134 cells/mm ± 0.28% .p = 0.01). The post hoc pairwise comparison showed that the mortality rate was significantly greater in group B compared to group A (p = 0.01).At T0, only the mortality rate was significantly different among the three groups , in Group A the mean ± SD ECD we found was 2040 ± 114 cells/mmp > 0.05).At T72, no significant difference in ECD, mortality rate or uncovered areas could be identified . Likewise, no difference was found in Overall Uncovered Areas (p = 0.1). In group C, Overall Mortality (7.66 ± 4.95%) and Total ECL (7.84 ± 4.94%) were higher than in group A (4.07 ± 3.76% and 4.37 ± 3.86%) and group B (3.97 ± 1.97% and 5.32 ± 2.90%). However, these differences were not statistically significant (p = 0.25 and p = 0.39).The mean ± SD ECD variation between T0 and T72 was 5.51 ± 1.79%, 3.06 ± 2.80% and 2.82 ± 2.58% in Group A, B and C, respectively  compared to that seen in Group B (2–8 °C) and A (>8 °C), although these differences did not reach statistical significance.Along with preservation time, the temperature is a crucial parameter for appropriate corneal tissue preservation, especially when the isolated Descemet Membrane, and its endothelium, are preserved . The finTo date, two main storage systems of corneal tissue have been developed: cold storage, mainly used in America and Asia, maintains the cornea between 2 °C and 8 °C for a maximum of 14 days, while Organ Culture preservation, which uses room temperature up to 31–37 °C for storage of corneas, can preserve the tissues for up to 28 days ,23. The In this study, the preservation of corneal tissue was performed in Optisol-GS, the most widely employed medium for CS . Other s2 has been associated with a significantly lower 5-year graft survival rate in a study by Vasiliauskaitė and colleagues [As DMEK continues to evolve, optimization of the preparatory steps leading to surgery will represent more and more a key area for development. The efforts to minimize ECL in the preparation, storing and shipping of preloaded grafts are paramount given its prominent role in early postoperative ECL. In fact, preoperative ECD has been directly correlated with 6-month post-DMEK ECD, which in turn was found to be a key predictor of late endothelial failure. Specifically, a 6-month ECD count of less than ~830 cells/mmlleagues . Given tlleagues ,3, the alleagues .Limitations of this study could be attributed to damage to the endothelium not related to hypothermic storage, which could have confounded our results. In the pre-loaded, ENDO-OUT configuration, the membrane is left free to roll up and then vacuumed through a tubing system connected to a syringe and can be accommodated in a larger space as compared to the devices for ENDO-IN membranes. Since the graft spontaneously rolls up with the endothelium folded outwards, corneal endothelial cells may be exposed to a certain degree of friction against the internal walls of the injector, especially during the injection phase when the diameter of the funnel is reduced . FurtherFinally, given the small sample size available, we might have been underpowered to detect significant differences among the three groups (Post Hoc Power range = 0.1–0.85).In conclusion, to preserve the quality of isolated DMEK grafts folded in the ENDO-OUT configuration in the DMEK RAPID Mini device in Optisol-GS, it is recommended to keep the grafts at temperatures between 2 to 8 °C. Lower temperatures can alter the morphological characteristics of the endothelium and possibly increase cell mortality. Conversely, no major issue has been observed with higher temperatures as long as these are kept below room temperature to avoid fungal growth .