Phospholipids with a choline head group are an abundant component of cellular membranes and are involved in many important biological functions. For studies on the cell biology and metabolism of these lipids, traceable analogues where propargylcholine replaces the choline head group have proven useful. We present a novel method to analyze propargylcholine phospholipids by MS. The routine employs 1-radyl-2-lyso-sn-glycero-3-phosphopropargylcholines as labeled lysophosphatidylcholine precursors, which upon cellular conversion direct the traceable tag with superb specificity and efficiency to the primary target lipid class. Using azidopalmitate as a click-chemistry reporter, we introduce a highly specific, sensitive, and robust MS detection procedure for the propargylcholine phospholipids. In a first study, we apply the new technique to investigate choline phospholipid metabolism in brain endothelial cells. These experiments reveal differences in the metabolism of phosphatidylcholine and its pendant, ether phosphatidylcholine. The novel method described here opens a new, quantitative, and detailed view on propargylcholine phospholipid metabolism and will greatly facilitate future studies on choline phospholipid metabolism. Phospholipids containing a choline moiety in their head group represent a major fraction of the cellular lipidome. The family of choline phospholipids includes phosphatidylcholine (PC), ether phosphatidylcholine (PC O), and sphingomyelin (SM). In most eukaryotic cells, PC comprises almost half of all phospholipids, whereas the generally less abundant SM or PC O show highly elevated levels in particular cells, e.g., in the brain or the heart.For investigations on the cell biology of choline phospholipids, an analogue of choline, propargylcholine, bearing a terminal alkyne moiety, was introduced . Upon meA particular strength of this tracer is the fact that the obtained localization data can be correlated with metabolic analyses. Propargylcholine phospholipid metabolism can be followed by TLC using fluorogenic reporter azides , 5 or byWe have recently introduced a highly sensitive MS method for tracing alkyne-labeled lipids employing a dedicated azide reporter that upon click reaction facilitates the ionization and identification of the labeled product . Using t3Pal) as a clickable MS reporter that allows for direct identification of the labeled propargyl phospholipids at the MS1 level by conferring a predictable mass shift to the analyte. Importantly, in negative ion mode at the MS2 level, a diagnostic fragment is formed that confirms the lipid identity while the individual side chains of the lipid are revealed in parallel. We have used this novel method in a series of experiments where we investigated the propargyl phospholipid metabolism in a brain endothelial cell line. This study opens a detailed and quantitative view on phosphocholine lipid homeostasis in bEND3 cells and demonstrates differences between regular and ether PC metabolism.Here, we demonstrate the applicability of our C171-based method for analyzing also head group-labeled alkyne lipids, the propargyl phospholipids. As the different positioning of the alkyne label at the head group imposes some intrinsic restrictions, we furthermore present a novel method overcoming these limitations. We therefore introduce azidopalmitate  in acetate buffer . After vigorous stirring at 30°C for 24 h, the organic phase was separated, and the solvent evaporated. The residue was separated by silica column chromatography (CHCl3/methanol/water 65/25/2) to yield 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine, propargyl phosphatidylcholine (pPC) O-16:0/18:1. Crotalus atrox snake venom was dissolved in borate buffer  and combined with a solution of pPC O-16:0/18:1 in diethylether/methanol 49/1. After vigorous stirring at 30°C for 4 h, the solvents were evaporated. The residue was separated by silica column chromatography (CHCl3/methanol/water 65/35/8) to yield LpPC O-16:0. The synthetic pPC 31:1 used as internal standard for MS analysis was synthesized from PC 31:1  was synthesized as follows. 16-Bromohexadecanoic acid  was stirred with sodium azide  in dimethyl sulfoxide for 24 h. Hexane/ethylacetate 3/1 was added to the reaction mix before several extractions with water. The organic phase was separated, and the solvent evaporated. Pure N3Pal was crystallized from hexane/ethylacetate 3/1. The MS-reporter azide (C171) has recently been described  and maintained in DMEM medium  containing 10% fetal calf serum  and 1% penicillin/streptomycin . Propargylcholine lipids were added to the medium at concentrations of 20 μM from 5 to 10 mM stock solutions in 80% ethanol. Cells were then cultured for 24 h.3 5/1 containing 240 pmol pPC 31:1, 210 pmol phosphatidylethanolamine (PE) 31:1, 396 pmol PC 31:1, 98 pmol phosphatidylserine (PS) 31:1, 56 pmol phosphatidic acid 31:1, 51 pmol phosphatidylglycerol 28:0, 39 pmol lysophosphatidate 17:0, 35 pmol lysophosphatidylcholine; 17:1, 38 pmol lysophosphatidylethanolamine (LPE) 17:1, 32 pmol ceramide 17:0, 99 pmol SM 17:0, 55 pmol glucosylceramide 12:0, 339.7 pmol triglyceride (TG) 50:1, 111 pmol cholesteryl ester 17:1, 64 pmol diglyceride 31:1, and 103 pmol monoglyceride 17:1 as internal standard , and redissolved in 10 μL CHCl3, and the tubes briefly vortexed. To tubes clicked with C171, 40 μl of C171 click mix were added (prepared by mixing 10 μl of 100 mM C171 in 50% methanol with 200 μl 5 mM Cu(I)AcCN4BF4 in acetonitrile [AcCN] and 800 μl ethanol) while tubes clicked with N3Pal, 70 μl of N3Pal click mix were added AcCN4BF4 in AcCN and 750 μl ethanol) followed by sonication for 5 min and incubation at 42°C for 16 h. About 300 μl of CHCl3 and 700 μl water were added, and samples were briefly shaken and centrifuged for 5 min at 20,000 g. The upper phase was removed, and the lower phase dried in a speed vac as above. About 500 μl of spray buffer (2-propanol/methanol/water 8/5/1 + 10 mM ammonium acetate) was added, and the tubes were sonicated for 5 min and stored at −20°C.Cells on 24-well dishes  were wasa), and to further six samples (sample b), another 250 pmol of all synthetic pPCs was added prior to the click reaction. All samples were click reacted with N3Pal before processing continued as usual. Samples were analyzed by MS, and the signal intensities of the homoacyl-pPCs were determined. Recovery as percentage was calculated from the signals according to 100 ∗ a/(b − a).Total lipids from 45,000 unlabeled bEND3 cells  were isolated as above but using an extraction mix also containing 250 pmol of synthetic pPC 18:0/18:0, pPC 18:1/18:1, pPC 18:2/18:2, pPC 18:3/18:3, pPC 20:4/20:4, and pPC 22:6/22:6. Six samples were processed as usual , its diagnostic fragmentation peak upon neutral loss (NL) of 335.26, and of the respective FA were recorded. Five replicate experiments were performed.Total lipids from 45,000 unlabeled bEND3 cells were isolated as above and mixed with increasing concentrations of synthetic homoacyl-pPCs and 240 pmol of pPC 31:1. Samples were processed as usual, and employing the Nm/z windows from 250 to 1,200 m/z (positive mode) and 950–1,300 m/z (negative mode) followed by recording MS/MS spectra  by data-independent acquisition in 1 m/z windows from 200 to 1,200 m/z (positive mode) and 950–1,300 m/z (negative mode).The tubes were sonicated for 5 min, and the dissolved lipids were analyzed. Mass spectra were recorded on a Thermo Q-Exactive Plus spectrometer equipped with a standard heated ESI source using direct injection from a Hamilton syringe driven by a syringe pump under the control of the Tune instrument control software. MS1 spectra  were recorded in 100 Raw files were converted to mzml files using MSConvert and analyzed using LipidXplorer . For ideP values were calculated.Statistical differences between sample groups were calculated using GraphPad Prism, version 8.0, software. Two-way ANOVA was followed by Dunnet analysis to correct for multiple comparisons. Family wise significance and confidence level  settings were applied. Multiplicity-adjusted To establish an improved method for choline phospholipid analysis by MS, we reasoned that a chemical modification of the propargylcholine moiety during sample preparation could facilitate the analysis of the lipid. Benefiting from the possibilities of the click reaction , 3, we fm/z 208.07  and heptadec-9-enoic acid (FA 17:1) side chains A. Upon cdetected D.Fig. 1ATo deepen the analysis, an identification of the individual FA side chains would be desirable. To detect fragments of the individual side chains generated in a MS2 setup, we opted for negative-mode MS. Consequently, the use of a different reporter for the click reaction became necessary. Such a reporter should include an azido moiety, ensure adequate ionization of the labeled product to enhance its signal, confer a predictable mass shift to the lipid analyte that allows for its direct identification at the MS1 level, and yield a diagnostic fragmentation pattern at the MS2 level.3Pal fulfills these requirements (m/z 227.20 and 267.23), revealing the identity of the attached FAs  can be detected along another head group-specific fragment (m/z 379.34). This way various lipid classes containing the propargylcholine head group can be analyzed . When analyzing the abundance of each FA among all 70 detected pPC subspecies, the generally most frequent palmitate, oleate, palmitoylate, and stearate ranked highest, and together comprised 83.4% of all FAs  appears to play a secondary role. However, certain flexibility for gross adjustments in head group composition may exist as we also find reductions in ethanolamine-containing lipids (endogenous LPE or PE) and PS for either precursor treatment. All other tested lipid classes displayed no changes with the notable exception of TG and cholesteryl ester, the relevance of the latter remains unclear.As the cells take up and metabolize these precursors, they exert parallel adjustments to their lipidome. Unsurprisingly, the pool of endogenous PC is affected strongest and reduced accordingly . The presn-1 position in membrane lipids. Hence, acylation of the LpPC 16:0 tracer would right away yield a very common lipid species, and yet, the cells spend considerable efforts to further modify at least 32% of these initial metabolites during remodeling. That way the cells maintain a specific side-chain composition within each lipid class that is paralleled by a certain head group distribution within the whole lipidome. Noteworthy, the profile of labeled pPC species observed in our experiments does not indicate whether it originates from head group or side-chain remodeling. Likely, both activities exerted by phospholipases C/D or A1/A2, respectively, will occur within our extended experimental time frame.Apart from the head group composition, cells are also known to precisely fine tune the FA profile within their lipidome. When comparing the labeled metabolites of LpPC 16:0 or LpPC O-16:0 to the endogenous lipid pool, we find a well-matching pattern of side-chain length and saturation. Not only similar species were identified but also the ranking of their abundance was similar. Although both tracers contain a saturated tail of 16 carbons, a great variety of side chains were found in the metabolites. For labeled pPC, a third of all species was not containing palmitate, and hence surely derived from lipid remodeling. While this again demonstrates the great acceptance of the propargyl label by the involved enzymatic machinery, it also shows the extent of lipid remodeling. Palmitate is generally considered the most abundant saturated FA and is usually found at the Comparing the remodeling of LpPC 16:0-derived and LpPC O-16:0-derived lipids, some differences in ether versus nonether lipids became apparent. When labeling cells under equal conditions, both precursors labeled their primary target lipid class with a superb efficiency of ∼85%. However, the total amount of metabolites from LpPC 16:0 was twofold lower than that from LpPC O-16:0. Conversely, the pool size of endogenous PC in bEND3 cells is tenfold higher than that of its ether pendant PC O. This led to very different proportions of labeled versus endogenous lipids in both classes. While a quarter of all PC molecules became labeled, one endogenous PC O was matched by three labeled PC O molecules. If assuming a similar rate of precursor uptake and primary acylation, a longer dwell time of the propargyl label within the ether versus the nonether PC pool is indicated. This is in accordance with the calculated long half-lives of ether lipids in neuronal cells and whole brain , 16 but When analyzing the label transfer away from the primary metabolite pool to other lipid classes, a comparable overall frequency (15% for pPC vs. 17% for pPC O) of head group exchange was found. However, some differences were observed. Head group transfer yielding labeled pSM was threefold more frequent for labeled pPC than pPC O, despite the latter being twofold more abundant. This may indicate that the involved enzymes, such as the SM synthases , 19, preTaken together, the biological data presented here open a quantitative view on the choline phospholipid metabolism in bEND3 cells. They reveal differences in metabolism of PC and ether PC while demonstrating the power of the newly introduced tracing tools. The novel method developed here will greatly facilitate further studies in the field.Data are available from the authors on reasonable request.The authors declare that they have no conflicts of interest with the contents of this article.


Additionally, enhanced contents of H2O2, O2−, and –OH scavenging rate; increased malondialdehyde (MDA) content; and transformation of unsaturated fatty acids (USFA) to saturated fatty acids (USFA) and reduced USFA/SFA (U/S) could result in lipid peroxidation and membrane integrity loss. Moreover, correlation analysis showed that lignin accumulation was in close relation to energy metabolism and lipid peroxidation in juice sacs of postharvest pumelos. These results gave evident credence for the involvement of energy metabolism and lipid peroxidation in the lignin accumulation of HR pumelo fruit during postharvest storage.Lignification is especially prominent in postharvest pumelo fruit, which greatly impairs their attractiveness and commercial value. This study investigated the energy metabolism and lipid peroxidation and their relationship with accumulated lignin content in juice sacs of “Hongroumiyou” (HR) during 90 d of storage at 25 °C. The results indicated that, the alterations of energy metabolism in juice of sacs of postharvest pumelos was featured by a continuous decline in energy charge and ATP/ADP; an increase in succinic dehydrogenase (SDH) activity before 30 d and increases in activities of cytochrome c oxidase (CCO) and F Citrus maxima (Burm.) Merr.), a citrus fruit of the family Rutaceae, is native to Asia and cultivated largely in China, Southeast Asia, and southern Japan [Pumelo , succinic dehydrogenase (SDH), ATPase, nicotinamide adenine dinucleotide phosphate (NADP), and nicotinamide adenine dinucleotide (NAD). ATPase plays a pivotal role in regulating substance transport, ATP synthesis, and cellular homeostasis [0F1-ATPase is critical in providing energy via oxidative phosphorylation and converting the energy of protons transported across membrane to synthesize ATP [1 to succinate [2, where proton gradient formed to promote ATP synthesis [Energy metabolism, which plays a role in postharvest crops, has been extensively reported ,8,9,10. eostasis ,18,29. Fsize ATP . A previsize ATP . SDH is uccinate . CCO is ynthesis ,34. Therynthesis . Qualityynthesis ,29,36. HFruit ripening and senescence is accompanied by disorder of ROS metabolism, resulting in excessive accumulation of ROS and malondialdehyde (MDA), which result in continuous lipid peroxidation and membrane injury, which consequently accelerate the quality deterioration of postharvest longan fruit , kiwifru0F1-ATPase, H+-ATPase and Ca2+-ATPase, contents of H2O2, O2−, and MDA, –OH scavenging rate, the relative contents of unsaturated fatty acids (USFA) and saturated fatty acids (SFA), and USFA/SFA (U/S) were analyzed in juice sacs of HR pumelos stored for 90 d. In addition, combined with correlation analysis, we proposed a probable mechanism of lignin accumulation associated with energy metabolism and lipid peroxidation in postharvest pumelos during storage.The current study aimed to illustrate the relationship of energy metabolism and lipid peroxidation with lignin accumulation in juice sacs of postharvest “Hongroumiyou” (HR) pumelos during storage. The EC, ATP/ADP, levels of NAD and NADP, activities of CCO, SDH, FHR pumelos were harvested according to our previous work  from an ATP, ADP, and AMP levels were measured according to the previous study . EC was g at 4 °C for 10 min. After neutralized with 0.1 M HCl, the supernatants were subjected to centrifugation at 10,000× g at 4 °C for 10 min. Then the supernatants were preserved on ice for further analysis. The enzymatic reaction was performed at 37 °C for 40 min with alcohol dehydrogenase and glucose-6-phosphate dehydrogenase as substrates for NAD and NADP detection, respectively. After dissolved in 1 mL 95% ethanol, the sample was detected at absorbance of 570 nm. The unit of the content of NAD and NADP was nM g−1 based upon fresh weight (FW) of juice sacs of pumelo fruit.Detection of NAD and NADP contents were on the basis of the method of Lin et al. , one gra0F1-ATPase activities. The SDH and CCO activities were detected on the basis of the method of Li et al. [0F1-ATPase was detected by using the F0F1-ATPase Assay Kit  following the manufacturer’s instructions. Definition of one U for F0F1-ATPase activity was as the enzyme quantity that produced 1 nM NADPH in one min of 1 g fresh tissue. The SDH, CCO, and F0F1-ATPase activities were described as U g−1 FW.Powdered juice sacs tissues (5 g) were employed for analyzing SDH, CCO, and Fi et al. . Definit+-ATPase in mitochondria and Ca2+-ATPase in plant cell basing on the published method of Jin et al. [+-ATPase and Ca2+-ATPase activities were as the enzyme quantity that catalyzed the production of 1 μM phosphorus in one hour at 660 nm of 1 g fresh tissue. The H+-ATPase and Ca2+-ATPase activities were described as U g-1 FW.Five grams of juice sacs of pumelo fruit were taken to analyze the activities of Hn et al.  and methn et al. , respect2O2 content, O2− content, and −OH. scavenging rate were measured using 5 g, 2 g, and 1 g of powdered juice sacs of pumelo fruit according to our previous work, respectively [2O2 content was described as μM g−1 FW, O2− content was described as nM g−1 FW, and –OH scavenging rate was expressed as %. MDA content was determined according to our previous study [−1 FW.The Hectively , the H2Ous study  with 1 gv/v = 1:1), followed by incubation at 60 °C for 60 min in the oven. After centrifuging at 5000× g for 5 min, a 0.22 μm membrane was used to filter the supernatant. One μL filtrate was subjected to gas chromatograph  according to the method illustrated in the previous study [18:1 + C18:2 + C18:3)/(C16:0 + C18:0).Relative amounts of fatty acids were determined according to our previous work . Then, 1us study . Respectp < 0.05). The correlationships were performed using excel software  via Pearson’s correlation analysis.All data was displayed as the mean ± standard error (SE). Statistical analyses were performed using SPSS version 26  via one-way analysis of variance (ANOVA) and Duncan’s test was used to test the significance of the difference  and ATP/ADP . Moreover, NAD and NADP, the primary types of pyridine nucleotide, were also associated with the energy level of harvested crops [2 = 0.6702, y = 2.2177x + 9.1611)  C. These  9.1611)  was corr2 = 0.815, y = 3.6293x + 16.109)   D. Additi 16.109) A exhibit 0.8112) E and ATP+ 2.8317 F from be0F1-ATPase activity increased firstly and then decreased after 60 d of storage  before 60 d of storage. Furthermore, the F0F1-ATPase activity     H and ATP+ 3.052) I before e energy . H+-ATPae of ATP . Ca2+-ATtructure . Abundantructure , litchi tructure ,57, broctructure , longan tructure . In our  storage A,B. Thesctively) J,K.+-ATPase and Ca2+-ATPase activities    L,M and Actively) A,B  N,O durin2O2, O2−, or –OH) are mainly generated from oxidative phosphorylation in the respiration cycle. ROS was reported to accumulate in various postharvest crops, such as grape [2O2 and O2− accelerate the peroxidation of membrane lipids, which might result in MDA accumulation and lead to fruit senescence [2O2 content increased as storage time processed       A, which  process . The con storage B. Correl 17.109) B. Meanwh 17.109) C. It is  2.9618) C, in add 4.2919) D, implyi 20.583) E. Intere 20.583) D. In our 20.583) , which w 20.583) . These r2O2 content (2O2 content (2O2 content (2O2 content (Cell membrane system is crucial in the normal physiological metabolism of fruit . Lipids  content F–H and b content I. Howeve content J,K, and  content C. Moreov content L–N, and  content O. Howeve content P,Q. Take0F1-ATPase activities before 60 d; but decreased H+-ATPase and Ca2+-ATPase activities. In addition, increased ROS level and MDA content induced the oxidation of USFA to SFA, resulting in lipid peroxidation and membrane integrity loss. These together may promote the lignin accumulation in postharvest HR pumelos during storage period. The probable mechanism of lignin accumulation in postharvest HR pumelos in association with energy metabolism and lipid peroxidation was shown in This study explored lignin accumulation in postharvest pumelos in association with energy metabolism and lipid peroxidation. In brief, the results displayed that the lignin accumulation in HR pumelos was closely associated with energy metabolism, which was mainly attributed to declined EC and ATP/ADP, increased SDH activity before 30 d, and increased CCO and F


Impacts in g forces were recorded throughout the race using the MARG sensor. Two blood samples were collected immediately pre- and post-race: serum creatinine (sCr) and albumin (sALB). (3) Results: Sixteen impact variables were grouped using principal component analysis in four different principal components (PC) that explained 90% of the total variance. The 4th PC predicted 24% of the percentage of change (∆%) of sCr and the 3rd PC predicted the ∆% of sALB by 23%. There were pre- and post-race large changes in sCr and sALB ( Acute kidney injury (AKI) is a relatively uncommon condition in sports. This condition has been reported in prolonged and repetitive strenuous exercises . It is uThe evidence of AKI cases in both contact and non-contact sports has been increased, but with clear different etiological backgrounds ,4,5. In In endurance running and mainly off-road running ,10, someMonitoring physical load is critical in endurance sports, such as off-road running, due to the high number of actions involved . This isParticipants were asked to perform three loops of a 12 km (+ascend = 600 m) circuit , under 25° Celsius of temperature, and 80% of humidity . Runners wore a MARG sensor attached to the lower back during the race, and variables of time-related impacts were extracted. Two blood samples were collected pre- and post-race to assess serum creatinine (sCr) and albumin (sALB). An analysis was made to explore a model based on impact variables that explained sCr and sALB increases between pre- and post-race.Eighteen experienced mountain runners participated in this study . They had 4.78 ± 2.42 years of experience competing in ultra-endurance events. Participant′s mean finish time was 4.2 ± 0.21 h. No neuromuscular, metabolic, or structural injuries were reported at least six months before the study. The participants were asked to avoid intense endurance exercise at least a week before the event.All participants were notified of the study′s aim, protocol details and the potential risks and rights during their participation. The study´s protocol followed all biomedical guidelines based on the Declaration of Helsinki (2013) and it was reviewed and approved by the Institutional Review Boards of Universidad Nacional (Reg. Code 2019-P005) and Universidad de Extremadura (Reg. Code 139/2020).n/min, g forces) variables were assessed using a Magnetic, Angular Rate and Gravity (MARG) sensor . The devices were attached to the lower back (~L1–L3) [Total/min) and fifteen progressively scaled categories of g-force magnitude, each 1 g wide (Impacts1–15 g/min).Sixteen different time-related impacts ((~L1–L3)  of each (~L1–L3) ,20, its (~L1–L3)  and its (~L1–L3) . The var®, Franklin Lakes, NJ, USA). After centrifugation (10 min at 2000 g), samples were stored at −20 °C. After 24 h, the samples were processed by the photometry method using an automatic biochemical analyzer . The variable analyzed was serum creatinine  and serum albumin . Acute kidney injury (sCr baseline in mg/dL *1.5) was considered following established diagnosis criteria [Blood serum samples were collected using 5 mL blood spray-coated silica tubes (BD Vacutainercriteria . Two groUrine specific gravity (USG) was assessed as a hydration status marker. USG was confirmed and double-checked with a digital valid  handheldKMO = 0.63) values and the Barleth Sphericity test (p < 0.01). Eigenvalues (EV) > 1 were considered for the extraction of each Principal Component (PC). A VariMax-orthogonal rotation method was used to identify the high correlation of components. A threshold of 0.6 was set to retain loadings. The highest loading was used when a cross-loading was found between PCs. PCA procedure followed standard quality criteria [All sixteen impact variables were grouped using a Principal Component Analysis (PCA) following previous studies guidelines ,26. PCA criteria , meetingt-test. The magnitude of the differences was calculated using Cohen´s d.A paired t-test was used to explore sCr and sALB changes between pre- and post-race data and the Change delta´s percentage (∆%) was calculated as follows: ((sCr post-race–sCr pre-race)/sCr pre-race)*100. An unpaired t-test was performed to explore potential differences in the number of impacts between those participants who met AKI diagnosis and those who did not. USG data were analyzed using a repeated measure 2R) was applied to resulted factor scores obtained from impact´s PCA using the ∆% of sCr and sALB as the dependent variable. This statistical technique was applied to identify which impact´s PC could predict the ∆% of sCr, and ∆% of sALB.Finally, a stepwise regression model .All variables were presented in mean ± standard deviation. Alpha was set at Participants experienced a total of 170.57 ± 34.42 impacts per minute. All sixteen impact-related variables were grouped in four different PC´s, explaining the 90.39% of total impacts cumulative variance. The 1st PC explained the 50.5% (EV = 8.08) of total variance, 2nd PC the 17.58% (EV = 2.81), 3rd PC the 13.05% (EV = 2.09), and 4th PC the 9.27% (EV = 1.48). Grouped variables and loadings are presented in t = −2.42, p = 0.03, d = −1.45, large effect size) but higher number of impacts in the 14–15 g category   registered lower number of impacts in the 1–2 g category (ize) see .. No dift = −6.24, p < 0.01, d = −1.47, large effect size) between sCr pre-race (1.24 ± 0.28 mg/dL) and sCr post-race (1.74 ± 0.41 mg/dL), and large differences  in sALB pre-race (4.33 ± 1.29 IU/L) vs. post-race (5.01 ± 0.86 IU/L). The ∆% of sCr was predicted by the 4th PC in a 24%  and the ∆% of sALB by a 23% . Finally, USG as a hydration marker reported no differences between pre- and post-race measurements .There were large statistical differences  and produce kidney damage compared to similar heavy muscular exercise, but without the massive g-forces. Consequently, it should be explored if downhill running, sudden change of direction, falls, or other similar high magnitude actions produce greater damage than other running actions . Finally, there is a need to use other blood markers  that allow researchers to differentiate AKI´s and extreme muscular exercise´s signs and symptoms. There is a need to review AKI′s diagnosis criteria and its validity when applying it to sport sciences and medicine.The results suggest that the magnitude and volume of running g-forces monitored with a MARG sensor attached to the lower back of off-road runners could predict the 24% change of serum creatinine and 23% change in serum albumin. These results must be confirmed in future research comparing similar heavy exercise with lower shock loads to the back and kidneys. Although these results may appear promising regarding the potential use of wearable devices to monitor cumulative mechanical kidney trauma in the future, greater understanding is required in the interaction of internal load  and external load  during prolonged exposure to vigorous repetitive exercise.The results suggest that a decrease in the amount and magnitude of impacts throughout a session or between sessions can be a way to mitigate the possible collateral damage of acute kidney damage during off-road running. The foregoing considers, therefore, that the monitoring and control of training external and internal loads is essential for the prevention and recovery of AKI in off-road runners. In this sense, it is essential to provide constant feedback on running loads behavior and wearable MARG sensors could be used for these purposes.


Artemia). The first available conversion models for syngnathids are provided, enabling their application to isotopic studies in the field and in the laboratory.Stable isotope analysis (SIA) was used to assess the influence of various preservation methods  on syngnathid (seahorses and pipefishes) fins, seahorse newborns (seahorses), and prey , seahorse newborns (three seahorses species), and prey (copepods and different stages of Artemia) commonly used to feed the fishes under rearing conditions. The aims of the study were: (i) to evaluate isotopic effects of chemical preservation methods across different types of organisms and tissues, using frozen samples as controls, and (ii) to construct the first conversion models available in syngnathid fishes. The chemical preservation in ethanol and, to a lesser extent, in formaldehyde significantly affected δ13C values, whereas the effects on δ15N signatures were negligible. Due to their low lipid content, the isotopic signals in fish fins was almost unaffected, supporting the suitability of dorsal fins as the most convenient material in isotopic studies on vulnerable species such as syngnathids. The regression equations provided resulted convenient for the successful conversion of δ13C between preservation treatments. Our results indicate that the normalization of δ15N signatures in preserved samples is unnecessary. The conversion models should be applicable in isotopic field studies, laboratory experiments, and specimens of historical collections.Isotopic stable analysis (SIA) is a powerful tool in the assessment of different types of ecological and physiological studies. For that, different preservation methods for sampled materials are commonly used prior to isotopic analysis. The effects of various preservation methods  were analyzed for C:N, and δ IsotWhenever possible, SIA is performed on muscle tissue due to its relative isotopic stability . Where lThe methods commonly used for tissue preservation include drying, freezing, ethanol or formaldehyde, depending on the objectives, limitations of the study and sampling conditions ,27,28,2913C when compared to other biochemical compounds , affecting bulk tissue signatures [15N values of the lipid-free sample [Lipids are depleted  in δgnatures ,30,31,32e sample . Alternae sample ,35. Idea13C and δ15N signatures both in syngnathid fishes, including adults (fin clipping) and newborns (bulk specimens), and in prey (copepods and different stages of Artemia) commonly used to raise those fishes. Syngnathids are a family of fishes including endangered and vulnerable species [13C and δ15N in syngnathid species considering different types of specimens and preservation methods. The models provided would be helpful in field, experimental and natural history collections.In the present study, we assessed the effects of freezing and solvent preservatives  on C:N ratios and δ species ,37. For  species . Since tAcartia tonsa) and Artemia . Microalgae  were cultivated at 22 ± 1 °C on F2P media to feed copepods Acartia tonsa and Artemia until the adult stage. The copepods were fed on the microalgae R. lens . Only copepods retained on a 180 µm mesh (copepodites and adults) were analyzed.We analyzed different sources of prey commonly used in the rearing of syngnathids: calanoid copepods  were hatched at 28 °C and newly hatched nauplii were collected to produce enriched metanauplii (from AF cysts) and adults (from EG cysts). For metanauplii, the nauplii were enriched (2–3 days at 100 Artemia mL−1) twice daily on a mixture including live microalgae P. tricornutum, Red Pepper  and dried Spirulina.Artemia was carried out at 26–28 °C. The adults were long-time enriched (3–6 days) or unenriched. The enrichment was carried out as previously reported [The production of adult reported .All samples were rinsed with distilled water, preserved according to the established procedures (see below), dried for 48 h (60 °C) and manually homogenized using a mortar and pestle.Syngnathus acus Linnaeus, 1758 and Syngnathus typhle Linnaeus, 1758, and seahorses Hippocampus guttulatus Cuvier, 1829, Hippocampus hippocampus Linnaeus, 1758 and Hippocampus reidi Ginsburg, 1933. The pipefishes were captured in Arcade Cove  in March–April 2016, transferred to the laboratory and fin clipped for further sampling. The seahorses were reared in captivity and sampled for dorsal fin tissue (fin clipping) and bulk newborn. The adults were fed on mixtures of adult Artemia and mysidaceans .A variable number (n > 5 per sample) of bulk juveniles were sampled for C:N and stable isotope analysis (SIA), and pooled prior to conservation. Newborn seahorses were sampled after the male’s pouch release (prior to first feeding) and euthanized with Tricaine MS-222 (0.1 mg L2O)  for comparisons on C:N ratios, and δ13C and δ15N values. Samples included prey (see above) and dorsal fins in five species of adult syngnathids . Additionally, the bulk newborn of three seahorse species  were also collected and preserved and analyzed as for dorsal fins.Tissue samples were submitted to direct freezing at −80 °C (control) or preserved in 95% ethanol (EtOH) or 4% formaldehyde (CHArtemia), which affected isotopic signals in both fins and newborn. Consequently, samples tissues comprised a wide range in isotopic values  and C:N ratios, as shown in Seahorse breeders were fed on different types of prey  were analyzed at Servizos de Apoio á Investigación (SAI) of the University of A Coruña (Spain) [13C or 15N and R is the corresponding ratio of 13C/12C or 15N/14N. As part of an analytical batch run, a set of international reference materials for δ15N values  and δ13C values  were analyzed. The range of C:N ratios in sampled tissues (2.9–6.3) were within the range (0.4–6.9) of reference materials used. The precision (standard deviation) for the analysis of δ13C and δ15N of the laboratory standard (acetanilide) was ±0.15‰ . Standards were run every 10 biological samples. The isotopic analysis procedure fulfils the requirements of the ISO 9001 standard. The laboratory is submitted to annual intercalibration exercises .Samples for stable isotope analyses (SIA) were homogenized and aliquots were transferred to preweighted tin capsules  . The analyses were made on sub-samples of 0.5–1 mg dry weight biomass. δ (Spain) . The samp < 0.05. The datasets on C:N and isotopic values in prey and seahorses (fins and newborn) were submitted for a Shapiro-Wilk test to confirm the assumption that the data were normally distributed, followed by analysis of covariance (ANCOVA) with organisms as covariates [Artemia), syngnathid genera (Syngnathus and Hippocampus), or seahorse species , depending on the analysis performed. Adjusted group means were obtained after partialing out the effects of the covariate using the Effect package in R. When significant, differences of means were submitted to multiple mean comparisons [The analyses were performed with R v.3.6.1  and Statvariates . Covariaparisons . Least sAnimal capture, handling and sampling were conducted in compliance with all bioethics standards on animal experimentation of the Spanish Government  and the Regional Government Xunta de Galicia (REGA ES360570202001/15/FUN/BIOL.AN/MPO01 and ES360570202001/16/EDU-FOR07/MPO01).13C and from 1.6 to 11.1‰ for δ15N, with mean values of −18.7 ± 2.5‰ and 6.2 ± 3.6‰, respectively  was 3.7–6.1 (mean: 4.8 ± 0.8) in prey, 2.9–3.5 (3.1 ± 0.2) in fish fins and 3.5–3.9 (3.7 ± 0.1) in fish newborn. Isotopic values in prey ranged from −21.1 to −14.7‰ for δectively .2O) of experimental samples  differed significantly  in their effect on C:N values (p < 0.081) (p < 0.001) in EtOH-treated samples , whereas the effect of CH2O was minimal  (p > 0.05), except in newborn seahorses (Δ = 0.29) (p < 0.001)  . When co< 0.001) .Hippocampus (2.99) and Syngnathus (3.14) differed significantly (p = 0.001), even though the difference was small. In seahorse newborn, mean C:N values were similar (p = 0.506), ranging from 3.59 in H. guttulatus to 3.64 in H. abdominalis.Considering dorsal fins, adjusted means for C:N values in 13C in prey, fins and newborns was highly affected (p < 0.005) by chemical preservation (p < 0.001) and less (p = 0.031) affected, respectively. The isotopic signal increased (enriched) significantly (p < 0.001) in EtOH-treated samples , except in clipped fins (Δ = 0.2‰) (p = 0.192). The treatment with CH2O led to decreased δ13C signals in prey (Δ = 1.1‰) (p < 0.001) but not in fish tissues (Δ = 0.0‰ in fins and 0.7‰ in newborns) . Broadly, the effects of preservation methods in newborn seahorses were similar to those in fins.The signatures for δervation . The eff13C were −20.9‰ in copepods and Artemia nauplii, −20‰ to −19.5‰ in Artemia metanauplii and −15.1‰ in non-enriched adult Artemia, and −16.3‰ in adult enriched Artemia. Isotopic signals for 13C in fish fins were −16.3‰ in Hippocampus and −13.3‰ in Syngnathus. Adjusted means in seahorse newborns were −17.2‰ in H. guttulatus, −19.5‰ in H. abdominalis and −21.0‰ in H. reidi.Considering prey, adjusted means for δ15N values were negligible, especially in fins (Δ < 0.2‰) and newborns (Δ < 0.3‰) (p < 0.001) (2O).Chemical treatments performed similarly to frozen samples, and the effects on δ< 0.001) . The hig15N in prey ranged from 2.7‰ in copepods and 20‰ to −11.3‰ in Artemia nauplii. Dorsal fins in Hippocampus and Syngnathus fishes differed in isotopic adjusted means . Adjusted means for δ15N in newborns were 12.5‰ in H. abdominalis, 13.2‰ in H. guttulatus and 20.7‰ in H. reidi.Adjusted means for δ15N and δ13C values in tissues treated with chemical preservatives relative to freezing treatment are provided in 2O-preserved newborns (Adj R2 > 0.384), the models were highly significant . The main discrepancies between the original and corrected models occurred in C:N values, especially in EtOH–treated samples.The correction equations to account for changes of C:N, δ15N and δ13C values in fins and newborns for treatment correction. Overall, isotopic corrected lines for both chemical preservatives did not differ from the 1:1 line. Besides this, the slopes in most uncorrected and corrected linear models did not differ significantly, except for δ13C in prey and fins. On the contrary, regression intercepts were significantly different in many cases. However, the differences were highly reduced (<0.01) in δ15N models, and corrected and control values were statistically undistinguishable.Simple arithmetic corrections could be applied to δ2O) on isotopic signals (δ13C and δ15N) and C:N ratios in syngnathid fishes, including adults , and newborn juveniles, and in prey commonly used to feed marine fishes in rearing systems. Considering the preservation methods tested, EtOH and CH2O are polar and non-polar solvents frequently used as preservation chemicals. Formaldehyde hydrolyzes proteins and systematically affects δ13C signatures [2O-treated materials were more similar to control samples than those preserved in EtOH, especially for C:N values. Correcting C:N ratios and isotope signatures in chemically preserved tissues with derived correction equations revealed significant differences for C:N and δ13C values between treated and control (freezing) samples. The easiest, fastest and most economical procedure for SIA in adult syngnathids in laboratory experiments would be freezing of clipped-fin tissue, whereas EtOH (does not requires defatting) or CH2O-treatments would be recommended in the field to ensure better sample maintenance during sampling.The present study demonstrates for the first time the effects of freezing and two chemical preservation methods  with C:N values  in controls. Hence, lower C:N ratios in bulk tissues (fins) led to lower shifts in EtOH-treated samples . Corrections on C:N ratios were reliable in prey but ineffective in fish tissues due to the low ratio variability derived from chemical treatments.13C signals. The depletion in 13C varied depending on the type of tissue, but prey and newborn were mostly affected . Those changes would be concordant with tissue lipid content [As compared to ethanol, formaldehyde-treated materials provided small shifts and low variability, affecting especially C:N values  and δ content  and/or t content ,44.13C signatures in fins (Δ < 0.2‰) and newborns (Δ < 1.0‰), we advise application of further corrections in chemical preserved tissues. On the contrary, δ15N signatures in solvent preserved materials were small enough (Δ < 0.2‰ in fins and <0.3‰ in newborns) to correct the values. Interestingly, average δ15N-shifts in both isotopes were much lower than the estimated trophic enrichment factor of 3.4‰ in aquatic animals [15N between frozen and solvent preserved tissues will not affect data interpretation in food web studies for syngnathids.Considering the average shifts achieved in δ animals . Hence, The diverse tissues of an animal differ in turnover rates due to inter-tissue differences in isotopic fractionation ,47,48,49ex situ experimental studies [H. guttulatus and H. kuda). Clipped filaments provide enough biomass for DNA analysis [Fish fins are excellent tissues for isotopic assessment, especially in studies involving threatened or endangered species ,22. Fin  studies . Partialanalysis ,52,53 buanalysis  and both13C when compared to proteins and carbohydrates [δ13C of tissues is the loss of lipids and/or proteins, resulting in 13C enrichment [13C, especially in materials with high C:N values . However, other studies reported different results [There is a high availability of isotopic studies on the effects of preservation methods and lipid normalization procedures in fish tissues ,46,55,56hydrates . Consequ results ,44.13C isotope, lipid extraction might also result in deviations in the δ15N of the lipid-free tissue [15N than bulk tissues. Commonly, δ15N signals in bulk tissues increase with trophic levels of organisms [δ15Nprey < δ15Nfin > δ15Nnewborn). However, the preservation in EtOH did not affect δ15N signals, suggesting that lipids in all tissues analysed were highly 15N-depleted, and that storage in those solvents will not impair the reliability of the analyses.Even though lipids mainly affect the heavier e tissue ,57,58. Arganisms , as refl15N values might also result due to artifacts in the extraction of lipids [13C and δ15N values in dorsal fin and muscle tissue, and significant effects of lipid extraction on δ13C values in muscle and liver [Hippocampus spp.; 2.93–3.53 in Syngnathus spp.). Accordingly, fin clipped samples could be submitted to SIA without the need of previous lipid extraction nor further mathematical lipid corrections for δ15N, whereas the regression models given in 13C.Impaired δf lipids ,56. In tnd liver . The stuThe main aim of the present study was to provide practical mathematical models for the conversion of isotopic signals in preserved live prey and syngnathids. The regression equations provided would be useful in field studies when samples cannot be properly stored, requiring preservation until further analysis. In addition, in spite of further assessment on the effect of long-term storage of sampled tissues on isotopic signals , the mod13C signals and C:N ratios, but the effects of the former were consistent and predictable, and can be corrected. Conversely, the shifts in δ15N of chemically preserved tissues were small enough to be ignored. Hence, both solvents provided consistent and reliable results. The first conversion models for the mathematical correction of data across the tested preservatives were constructed specifically for syngnathids. Those taxa-specific models may be applied to field collected samples as well as to historical collections. Further work should be conducted to determine the isotopic effect of lipid extraction and duration of preservation, except in dorsal fins.The results achieved revealed different effects of chemical preservation on isotopic signatures and C:N ratios both in syngnathid fishes  and in prey commonly used to feed those fishes in the laboratory. Considering that dorsal fins are valid isotopic subrogates of muscle tissue in syngnathids, our results would be comparable to those in muscle tissue samples. However, the effects of feeding activity on potential differences between both tissue types deserve further consideration . The imp


Progressive vision loss in adults has become increasingly prevalent worldwide due to retinopathies associated with aging, genetics, and epigenetic factors that damage the retinal microvasculature. Insufficient supply of oxygen and/or nutrients upregulates factors such as vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), which can induce abnormal angiogenesis and damage the structural arrangement of the retinal blood barrier (BRB). Müller glia (MG) regulate the diffusion of essential compounds across the BRB and respond to retinal insults via reactive gliosis, which includes cell hypertrophy, migration, and/or proliferation near areas of elevated VEGF concentration. Increasing concentrations of exogenous VEGF, upregulated by retinal pigmented epithelium cells, and endogenous epidermal growth factor receptor (EGF-R) stimulation in MG, implicated in MG proliferative and migratory behavior, often lead to progressive and permanent vision loss. Our project examined the chemotactic responses of the rMC-1 cell line, a mammalian MG model, toward VEGF and EGF signaling fields in transwell assays, and within respective concentration gradient fields produced in the glia line (gLL) microfluidic system previously described by our group. rMC-1 receptor expression in defined ligand fields was also evaluated using quantitative polymerase chain reaction (qPCR) and immunocytochemical staining. Results illustrate dramatic increases in rMC-1 chemotactic responses towards EGF gradient fields after pre-treatment with VEGF. In addition, qPCR illustrated significant upregulation of EGF-R upon VEGF pre-treatment, which was higher than that induced by its cognate ligand, EGF. These results suggest interplay of molecular pathways between VEGF and EGF-R that have remained understudied in MG but are significant to the development of effective anti-VEGF treatments needed for a variety of retinopathies. Increasing numbers of adults worldwide are experiencing progressive and permanent loss of vision ,2. Many Breakdown of the BRB and occlusion of microcapillaries often leads to progressive retinal dysfunction, characterized by ischemia and/or hypoxia, i.e., diminished and/or insufficient levels of blood and oxygen in retinal tissue, respectively ,12. In rVEGF expression impacts multiple signaling pathways central to homeostasis and repair through cross-talk with Akt/PBK and MEK . The relThe current project examined the relationship between VEGF signaling and expression of EGF-R leading to rMC-1 chemotaxis, or directional migration, central to both neuroprotective and neurodisruptive glial responses . Control2. Two weeks prior to experiments, cells were cultured in serum-restricted media (1% FBS in DMEM) at a concentration of 1.0 × 106 cells/mL in T-75 flasks and passaged as needed. Cultured rMC-1 were dislodged using Accutase® Solution  and re-suspended in 10% FBS supplemented DMEM for testing.MG in this project were modeled by cultured rMC-1 cells , derived from rat primary MG cells and immortalized using SV40 T-antigen. These cells were selected because they express well-known markers characteristic of primary MG ,37,38,39Expression levels of EGF-R , VEGF-R , FGFR-2 (fibroblast growth factor 2 receptor), and FGFR-8 (fibroblast growth factor 8 receptor) were measured using qPCR, and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression, as previously described by our group . rMC-1 cAutomated PCR was performed in a final volume of 20 μL containing 5 ng of RNA template, 10 μL of TaqMan RT-PCR Mix, 1 μL of the Taqman Gene Expression Assay primer and 0.5 μL of Taqman RT Enzyme Mix in a StepOne Plus Real-Time PCR system . Reverse transcription was performed at 48 °C for 15 min, followed by denaturation of the cDNA and activation of the DNA polymerase at 95 °C for 10 min. A total of 40 cycles of 15 sec at 95 °C and 1 min at the annealing temperature of 60 °C were used to amplify the PCR product. The relative change in expression levels between the control and experimental conditions was calculated using the conventional ΔΔCT method, as used previously by our group ,40,41. P5 cells/mL, and approximately 0.5 mL of cell solution was added to each borosilicate glass chamber  with serum-free media, and left to adhere for 24 h. Media was removed and the samples were washed 3 times with Dulbecco’s phosphate-buffered saline  solution, fixed with 10% formalin  for 10 min then rinsed twice with PBS. The samples were permeabilized with 0.1% Triton X  in 0.1% Bovine Serum Albumin  in DPBS for 10 min and then blocked with 1.0% BSA in PBS for 1 h. The samples were rinsed twice with the same blocking solution prior to being exposed to a 1:200 dilution of either EGF-R antibody, rabbit monoclonal  or VEGF receptor 2 monoclonal antibody  in antibody diluent  overnight at room temperature (25 °C). The samples were washed 3 times with DPBS, exposed to a 1:200 dilution of goat anti-rabbit igG (H+L) secondary antibody  for 30 min, and then rinsed 3 times with DPBS. Nuclear staining  was performed for 20 min at room temperature (25 °C), then samples were washed again with DPBS and covered with glycerol  to prevent drying. All data were measured and compared to basal conditions in triplicate.rMC-1 were plated at a concentration of 2.5 × 105 cells/mL in serum-free DMEM on the top part of a two compartment transwell model, divided by an 8 µm porous polyester membrane of 10µm thickness , as per 2. Upon completion, the top surface of the membrane was cleaned with an applicator followed by fluorescent staining of the bottom surface of the membrane, containing the cells that migrated through it, using Cell TrackerTM Green CMFDA  at a concentration of 15 µM. Numbers of motile rMC-1 located at the bottom side of the porous membrane were optically measured for all tests. rMC-1 migration in response to ligand signaling through these transwell assays was examined using four conditions: (i) rMC-1 stimulated by EGF signaling fields; (ii) rMC-1 stimulated by VEGF signaling fields; (iii) rMC-1 exposed to VEGF for one hour and then stimulated by EGF signaling; and (iv) control . Each testing condition was examined in triplicate.Boyden chambers  in combination with thin porous membranes were used as transwell assays for this study. rMC-1 were seeded at a concentration of 2.5 × 10H), as per 3)is the solution concentration, t (seconds) denotes time, ū (m/s) is bulk velocity, D (m2/s) is the diffusion coefficient, and x denotes position within the microchannel length [The transport phenomena governing the development of concentration gradients in the glial line system (gLL), has been previously described by our group , and usel length .2 incubator. Excess PLL solutions within gLL interstitial spaces were then aspirated out and devices cleaned via manual PBS wash using a 1 mL syringe. A rMC-1 solution of 1 × 106 cells/mL was seeded into the sink reservoir and microchannel of gLL devices using a 1 mL syringe and left to adhere for 2–4 h. Solutions of EGF  or VEGF  were added to the gLL source reservoir using a micropipette and allowed to transport overnight along the adjoining microchannel towards the sink reservoir. The concentrations were selected as per previous results from our lab demonstrating the highly chemotactic response of rMC-1 cells towards these fields in comparison to other concentrations of these growth factors [Inner surfaces of multiple gLL devices were individually functionalized with Poly-L-Lysine (PLL) at a concentration of 15µg/mL  diluted in DPBS. A 100 µL volume of this extracellular substrate was loaded into the devices via syringe and allowed to crosslink at 37 °C overnight in a 5% CO factors . The dis factors ,43,44,45i,yi) and  are then added to determine the total path length or distance travelled by cells during testing.Cell migration in response to EGF or VEGF stimulus was examined using four testing conditions: (i) rMC-1 stimulated by EGF signaling fields; (ii) rMC-1 stimulated by VEGF signaling fields; (iii) rMC-1 exposed to VEGF for one hour and then stimulated by EGF signaling; (iv) rMC-1 exposed to EGF for one hour then stimulated by VEGF signaling; and (v) control . Live and adherent rMC-1 were imaged every 30 min for a total of 6 h to record the cell trajectories and total distances traveled in response to applied chemical stimulus. Only rMC-1 cells identified at time t = 0 were tracked for quantification of migration for each group, to avoid recording the path of cells that may have proliferated over the short time-span. We note, however, that our previous work ,33 indic−6 cm2/s for EGF [−7 cm2/s for VEGF using the Stokes–Einstein equation [Concentration gradients of VEGF in transwell assays and in the gLL were modeled in COMSOL Multiphysics 5.3a  using physical properties of VEGF and the true geometry of the assays, as per manufacturer references and our own measurements . Transport of EGF and VEGF within the gLL and transwell assay was computationally modeled to predict reagent concentration over time across the microchannel and permeable membrane, respectively. The diffusivity of EGF was estimated to be 2.0 × 10 for EGF , and 9.0equation ,48.An Inverted transmitted light microscope  was used to observe rMC-1 behavior over time and to perform optical analysis with a cooled CCD camera  of a 20× objective magnification . A ZEISS LSM 800 confocal microscope at 63× with an oil immerged objective was used for immunocytochemistry (ICC). Brightfield images of rMC-1 were evaluated using ImageJ (NIH imaging software). Immunostained rMC-1 images were assessed using ZEN Blue 2.5 software (ZEISS imaging software) to evaluate the fluorescence intensity.n = 10 to 15 cells per device, using 5–7 independent devices per experimental condition. Values are reported using mean and standard deviation. The post-hoc Tukey and Dunn with Holm correction tests were used to determine statistical significance between conditions, where p-values < 0.05 were denoted by an asterisk, *, and p < 0.01 were marked with a double asterisk, **.One-way ANOVA was used to analyze statistical significance among parametric data sets, whereas Kruskal–Wallis rank sum was used for non-parametric ones. Each data set was gathered from a minimum of The first set of experiments measured the effect of receptor upregulation in rMC-1 upon stimulation with EGF, VEGF, fibroblast growth factor 2 (FGF2), and fibroblast growth factor 8 (FGF8). Values were normalized against controls and shown in C = 0.27 ± 0.098 for control, IE = 0.37 ± 0.063 in response to EGF signaling, and IV = 0.46 ± 0.074 for rMC-1 stimulated with VEGF. Statistical significance was found between the EGF and VEGF-treated groups with respect to control (p < 0.01).We next examined the rMC-1 expression levels of EGF-R in response to both EGF and VEGF stimuli using immunocytochemical staining (ICC). As shown in TA, and in the gLL microfluidic system, VEGFgLL, were modeled over time. We note that CGPs for both ligands were produced, but only VEGF is shown for illustration. TA, decreases rapidly to produce very shallow concentration gradients that approached zero . In contrast, the distribution of VEGF within the gLL microfluidic system, VEGFgLL, slowly evolved over 48 h to produce quantitative changes in signaling fields. As shown, distinct, non-linear distributions of VEGF were produced along the channel length, l, at each time point from 6 h to 48 h. Further, each gradient profile in the gLL produced both shallow and steep gradients of VEGF at different spatial positions over time.We next used two different technology platforms to create extracellular gradients of the studied ligands in transwell assay (TA) and gLL platforms. Both systems were used because they each create different concentration gradient profiles and have been widely used in chemotactic studies of neural cells ,49,50. CTANC = 12.1 ± 4.23 µm. rMC-1 migration towards EGF signaling (E) in TA was measured as TANE = 36 ± 12.8 µm; migration towards VEGF signaling (V) was measured as TANV = 87.2 ± 25.8 µm. Numbers of motile rMC-1 responding to the mixed VEGF/EGF signaling condition (VE) were measured as TANVE = 77.1 ± 44.4 µm and rMC-1 stimulated to the mixed EGF/VEGF condition (EV) were measured as TANEV = 39.7 ± 17.7 µm. Assessment for data normality was performed using the Shapiro–Wilk test, which concluded the data did not fit a normal distribution. Hence, the Kruskal–Wallis rank sum test was used to evaluate statistical significance, and a Dunn post hoc test with Holm correction to discern significance among groups. TANV and TANVE demonstrated statistical significance against control (TANC). The corresponding p-values were found to be 0.0295, 3.206 × 10−8, 3.41 × 10−6 and 0.0145 for the groups TANE, TANV, TANVE and TANEV against control, respectively.Our study next examined rMC-1 migration within both TA and gLL microfluidic platforms. Migration was measured in response to EGF signing fields, VEGF signaling fields, and a mixed condition of VEGF-treated rMC-1 responding to EGF signaling fields. gLLPLC =14.2 ± 0.5.7 µm. As seen, rMC-1 migrated towards EGF (E) with an increased path length of gLLPLE = 39.7 ± 8.96 µm, and towards VEGF (V) with gLLPLV = 74.7 ± 17.7 µm. Motile rMC-1 in the mixed condition of VEGF/EGF stimuli (VE) produced an average value of gLLPLVE = 56.3 ± 6.64 µm, and the mixed condition of EGF/VEGF stimuli (EV), gLLPLEV = 28.96 ± 5.6 µm. Statistical significance was measured across all groups with respect to control: where ** symbolizes p < 0.01 and * for p < 0.05. Holm–Bonferroni method yielded p-values of 1.79 × 10−6, 0, 1.03 × 10−11 and 1.88 × 10−3 when compared to control for the groups: gLLPLE, gLLPLV,gLLPLVE and gLLPLEV, respectively. gLLMCC = 46.67% for control (C), gLLMCE = 93.33% for EGF stimulation, gLLMCV = 100% for VEGF stimulation and gLLMCVE = 93.33% for VEGF pre-treatment towards EGF. Standard error was between 3%–6% of the mean values in all cases. Note that MC values cannot be calculated for TA platforms since this test only provides the number of motile cells adhered to the membrane, and not the path length of their individual migration.Migration in the gLL system enabled study via average path length, PL, and percentage of motile cells, MC. The average distances traveled in response to signaling produced by the gLL microfluidic system are shown in The experiments performed in this project used cultured cells to model primary MG via the rMC-1 model , an immortalized MG cell line derived from adult rats. Although this cell line has been characterized for over a decade, recent research has characterized again their genotype to verify their nature, including T-antigen verification, gene expression and protein expression of molecules including GFAP, CRALBP and vimentin ,38. The VEGF is a key angiogenic molecule essential to both reparative and pathological processes in the visual system. While VEGF can promote cell viability and connectivity in retina ,57, it iEGFR upregulation in the presence of VEGF has been well studied in glial tumors ,65, wherOur study first examined gene expression of EGF-R in response to key factors associated with angiogenesis. Dramatic upregulation of EGF-R DNA was measured upon VEGF stimulation than that recorded in response to EGF, FGF2, or FGF8 stimulation via qPCR . These dMG cells in mammals respond to injury by proliferating, extending their processes beyond the outer limiting membrane, and migrating in later stages of retinal degeneration ,70. BreaMigration of rMC-1 in response to VEGF and EGF signaling was next evaluated using both transwell assays and our microfluidic assay, the gLL. TA, such as Boyden chambers, have been well utilized in chemotactic study to evaluate the chemotactic strength of different ligands and/or ligand combinations upon a variety of cell types ,76. HoweIn both platforms, VEGF induced much greater numbers, N, of motile rMC-1, as well as greater fractions of motile cells, MC, and larger migration distances, PL, as measured by the gLL. However, rMC-1 pre-treated with VEGF exhibited significantly higher N in the TA platform, as well as higher PL along EGF fields in the gLL device . This coTaken together, our study used quantitative platforms such as the gLL and transwell assays to point out the interplay between VEGF stimulation and subsequent EGF-R upregulation in rMC-1 chemotaxis. Results illustrate that treatment of VEGF in rMC-1 led to chemotactic responses, which could help to gain insight on the role of VEGF and the consequent reactive behavior in MG in DR. Further exploring signaling pathways that link EGF-R upregulation with elevated concentrations of VEGF will aid current development of retinal therapies.


Multipotent mesenchymal stromal/stem cell (MSC) therapy is under investigation in promising (pre-)clinical trials for wound healing, which is crucial for survival; however, the optimal cell dosage remains unknown. The aim was to investigate the efficacy of different low-to-high MSC dosages incorporated in a biodegradable collagen-based dermal regeneration template (DRT) Integra®.N = 8 Yorkshire pigs) and seeded between 200 and 2,000,000 cells/cm2 of umbilical cord mesenchymal stromal/stem cells on the DRT and grafted it onto full-thickness burn excised wounds. On day 28, comparisons were made between the different low-to-high cell dose groups, the acellular control, a burn wound, and healthy skin.We conducted a porcine study (2 regenerates the full-thickness burn excised wounds most efficaciously, followed by the middle dose range of 200,000–400,000 cells/cm2 and a high dose of 2,000,000 cells/cm2. The low dose of 40,000 cells/cm2 accelerated reepithelialization, reduced scarring, regenerated epidermal thickness superiorly, enhanced neovascularization, reduced fibrosis, and reduced type 1 and type 2 macrophages compared to other cell dosages and the acellular control.We found that the low dose range between 200 and 40,000 cells/cmThis regenerative cell therapy study using MSCs shows efficacy toward a low dose, which changes the paradigm that more cells lead to better wound healing outcome.The online version contains supplementary material available at 10.1186/s13287-020-02131-6. After a skin injury, skin regeneration and wound healing of the epidermis and dermis are crucial to lowering the risk of infections associated with high mortality . Therefo2 . The MSC-treated groups showed a median between 96 and 81% epithelialization compared to the acellular control with a median of 92% (IQR 89–95). The low dose group with 5000 cells/cm2 showed the fastest epithelialization with 96% epithelialization (IQR 91–97), followed by 40,000 cells/cm2 with 95% epithelialization (IQR 89–96). The lowest dose of 200 cells/cm2 and high doses of 200,000–2,000,000 cells/cm2 showed inferior wound healing compared to the acellular control with epithelialization between 81 and 91% (IQR 69–92) . The highest dose of 2,000,000 cells/cm2 (IQR 4–9) and the lowest dose of 200 cells/cm2 (IQR 5–9) both had the same median VSS of 6. The other MSC-treated groups of 5000, 200,000, and 400,000 cells/cm2 showed a median VSS of 8 , compared to the acellular control with the same median VSS of 8 (IQR 7–10). Overall the MSC-treated groups appeared less inflamed, with a more homogenous scar texture. The lowest and the highest dose had a sample size of N = 3, while the other dose groups had N = 6 , which is the most recognized and validated  scar sca 8 IQR 7–0. Overal2 with a median of 157 μm (IQR 99–198), followed by the dose of 40,000 cells/cm2 with a median of 189 μm (IQR 132–262), and the dose of 400,000 cells/cm2 with a median of 131 μm (IQR 116–149). The acellular control showed a median of 177 μm (IQR 64–383 μm), although it lagged in epidermal regeneration and demonstrated a high range of hypo- and hyperplastic epidermal thickness, where the Integra® scaffold was incompletely degraded by day 28. The DRT was visible in none of the MSC-treated groups. The dose of 5000 cells/cm2 showed a median of 284 μm (IQR 205–286 μm) and, according to the reference, was defined as hyperplasia, although the histology showed a very homogenous epidermal regenerated architecture with rete ridges comparable to the other MSC-treated groups , and burn wounds, without any treatment, had a median of 63 μm (IQR 49–75 μm). Hypo- and hyperplasia were defined as inferior or superior epidermal thickness from the interquartile range of the healthy skin. The best regenerated epidermal thickness was achieved from the dose of 200,000 cells/cm2 reached the highest collagen density , followed by 200,000 cells/cm2  and 5000 cells/cm2 . The acellular control reached the lowest collagen density   Figs. c and 3b.2 showed the same vessel count of 17 (IQR 12–17) compared to the healthy skin . Closest to the reference, the dose of 200,000 cells/cm2 showed 15 vessels (IQR 12–16), followed by 400,000 cells/cm2 with 19 vessels (IQR 15–23). The acellular control regenerated 7 vessels (IQR 6–10)  dose group, followed by 200,000 cells/cm2 , and 5000 cells/cm2   were stained and measured with imaging software after extraction and an adjusted density assessment , 47, 48.2 with a median of 8 (IQR 6–11), followed by 200 cells/cm2 with a median of 12 (IQR 5–15) and 5000 cells/cm2 with a median of 12 (IQR 7–18). The wounds with 400,000 cells/cm2 showed a median of 22 (IQR 21–35) and the wounds with 2,000,000 cells/cm2 showed a median of 37 (IQR 31–44), which was more compared to the acellular control with a median of 17 (IQR 10–30). Evaluating the dermal region, the dose of 40,000 cells/cm2 showed the lowest positive cell count of 7 (IQR 6–11), followed by the dose of 5000 cells/cm2 with a median of 9 (IQR 8–10), and 200 cells/cm2 with also a median of 9 (IQR 6–16). The acellular control showed a lower median of positive counted cells of 32 (IQR 15–49) compared to the highest dose group with 2,000,000 cells/cm2 with a median of 36 (IQR 8–50) , followed by 40,000 cells/cm2 with a median of 12 (IQR 8–24), and 200 cells/cm2 with a median of 25 (IQR 16–54), than the acellular control with a median of 68 (IQR 48–72). In the dermal part, all MSC-treated groups showed fewer positive cells than the acellular control with a median of 68 (IQR 34–72). Within the different dose groups, we found the lowest positive cell count when treating wounds with 5000 cells/cm2 with a median of 8 (IQR 6–13), followed by 40,000 cells/cm2 with a median of 12 (IQR 4–29), and 200,000 cells/cm2 with a median of 31 (IQR 25–34) . First,poptosis  and addipoptosis , 58–60, However, the explanation why different cell-dosages have varying efficacies might be more complex. The extracellular microenvironment  is taken into account. For instance, recent studies have shown that the collagen scaffold as MSC carrier leads to inferior wound healing compared to xenografts , but theThis analysis presented here evolved after an unexpected observed low-dose healing phenomenon in an ongoing trial, where we retrospectively analyzed our collected dataset. We therefore recommend for future research to create a dose-model that is translatable and to implement objective scientific methods to determine healing or any outcome measures of interest . This would have been interesting but not feasible in such large inflicted injuries primarily investigating wound healing (and therefore avoiding wound biopsies for proofing). Each cell manipulation can potentially affect the transplanted cells by inducing down-stream changes . We perf+-tracing to determine cell fate would have been interesting, but this was not our primary focus.In comparable cell tracing experiments in Integra®, it was shown that the cells were also no longer detectable , 34 afteFurthermore, it would have been interesting to take biopsies and investigate the molecular cytokine profile  and perf2. Additionally, the (extreme) lowest dose of 200 cells/cm2 and (extreme) highest dose of 2,000,000 cells/cm2 would have not been included in the model, due to the sample size. Therefore, we decided to show the descriptive data rather than the misleading GEE significance.For statistical analysis, we tried to cluster MSC doses in our non-parametric dataset to a low-middle-high dose, using generalized estimating equations (GEEs) models. This estimation model accounted for dependencies among the data introduced by multiple wounds on the same pig , including the treatments , which were performed on 3–7 different pigs, between 3 and 12 times. However, due to the low N the determination between the clusters would have neglected the third best results of 200,000 cells/cmOur results in this pre-clinical study highlight two directions for future research. First, the dose-model can be translated to humans for potential autologous MSCs treatment trials, due to the principal similar skin structure from pigs to humans. MSCs treatment works at any dosing—from low to high; however, it is crucial to determine the most optimal cell therapy for patients, for partial and full-thickness regeneration, for acute and chronic wound healing.Second, an off-the-shelf therapy using the released paracrine products will be successful, if the optimized “cell dose cocktail” is quantitatively determined. This therapy can then act as a frontier in regenerative medicine.This study gives new insights based on a cell-dose-dependent wound healing model in full-thickness skin regeneration and shows most efficacy in low doses compared to higher dosages. This is a decisive finding for future investigations using stromal/stem cells. To the best of our knowledge, there are no comparisons available that including such a wide range of doses in such a large pig animal trial. This cell-dose model can be translated and implemented to innovative, regenerative stem cell therapy.Additional file 1: Figure S1. Schematic purpose of the DRT Integra®, cell incorporation into the DRT, cell viability after labeling cells with DiO, DRT cell infiltration, DiO detection via flow cytometry, DRT Remodeling. (A) Schematic purpose of the DRT. Excision and removal of the wound tissue, grafting of the DRT for cell ingrowth and remodeling until the protecting silicon layer is removed. (B) Cellularization of the DRT. (1) Stained after 12 h with ActinGreen and DAPI, horizontal view. (2) Vertical view, green channel, stained with ActinGreen. (3) Seeding depth 123 ± 21 μm , in the 1.3 mm thick scaffold. (C) Live-Dead-Staining 12 h after flow cytometry and DiO-labeling, confocal microscope, magnification × 20, as followed:  Live cells  (green channel),  Dead cells (EthD) (red channel),  Merged. (D) H&E stained DRT after tissue biopsy and tissue preparation, on day 4 and 7, magnification × 20. Dark yellow colored line indicates the upper boarder from the DRT. The DRT is violet stained (as seen in both images on day 4). The brown line at the left bottom image border indicates the DRT scaffold structure. (E) Flow cytometry on day 7 after tissue preparation with a double positive cell signal of a cell surface dye (DIO) on CD90+ cells. (F) Masson’s Trichrome Staining of Histology on day 7, 14, 21 and 28. Magnification × 10. Figure S2. Line of best fit for cell dose concentration per parameter. Each graph illustrates a regression (of order 2) with the line-of-best-fit; x-axis is cell dose concentration shown on a logarithmic scale and on the y-axis are the parameters. The 95-confidence-interval is the area in light blue. . Table S1. Future directions, outlook and other research questions. Potential associated explanations for different outcome in the presented data set.