Studying astroglial K+ changes is, however, hampered by a lack of suitable techniques. Here, we combined results from fluorescence imaging, ion-selective microelectrodes, and patch-clamp recordings in murine neocortical slices with the calculation of astrocytic [K+]. Brief chemical ischemia caused a reversible ATP reduction and a transient depolarization of astrocytes. Moreover, astrocytic [Na+] increased by 24 mM and extracellular [Na+] decreased. Extracellular [K+] increased, followed by an undershoot during recovery. Feeding these data into the Goldman–Hodgkin–Katz equation revealed a baseline astroglial [K+] of 146 mM, an initial K+ loss by 43 mM upon chemical ischemia, and a transient K+ overshoot of 16 mM during recovery. It also disclosed a biphasic mismatch in astrocytic Na+/K+ balance, which was initially ameliorated, but later aggravated by accompanying changes in pH and bicarbonate, respectively. Altogether, our study predicts a loss of K+ from astrocytes upon chemical ischemia followed by a net gain. The overshooting K+ uptake will promote low extracellular K+ during recovery, likely exerting a neuroprotective effect. The resulting late cation/anion imbalance requires additional efflux of cations and/or influx of anions, the latter eventually driving delayed astrocyte swelling.Malfunction of astrocytic K By takiut 10 mM ,3,4. Thirization ,6. The mchannels ,8. Astroin [K+]o ,10.+]o by astrocytes is thus largely dependent on their NKA activity and on intact energy metabolism. In the core region of an ischemic stroke, failure of energy metabolism results in a breakdown of astrocyte K+ regulation and [K+]o homeostasis o and promoting neuronal depolarization during energy failure [+ as well as Ca2+ concentrations [+]o, suggesting that a disturbance in astrocytic K+ regulation represents an important initial event i during energy failure are extremely rare [+]o o . A limits exists ,24,25,26+]i mathematically, exploiting the dominating plasma membrane permeability of astrocytes for K+  equation. Notably, we determined all parameters required experimentally, namely [K+]o, intracellular and extracellular Na+ concentration , membrane potential (EM), and relative Na+ over K+ permeability (α). This allowed us to estimate astrocytic [K+]i, as precisely as possible, significantly extending former studies using this strategy.As an alternative to direct measurement, former studies approached astroglial ”).+]i, tissue slices were bolus-loaded with the membrane-permeable form of SBFI . SBFI was excited at 400 nm and its fluorescence detected above ~430 nm (409 beam splitter and 510/84 emission filter)  and pH units, respectively, using established in situ calibration procedures i of layer II/III neocortical astrocytes was determined recently in our laboratory employing an approach introduced by Mondragao et al. o, [Na+]o, and extracellular pH (pHo) were measured in acute tissue slices using double-barreled ion-sensitive microelectrodes. These were prepared from two thin-walled borosilicate glass capillaries with filament , glued and pulled out together as described before  was 0–10 mM and [Na+] adjusted accordingly. Na+-sensitive electrodes were calibrated in salines composed of 25 mM HEPES, 3 mM KCl, and a total of 160 mM NaCl and N-methyl-d-glucamine chloride (NMDG-Cl), with [Na+] ranging from 70–160 mM and [NMDG+] adjusted to maintain osmolarity. Calibration of pH-sensitive microelectrodes was conducted in salines at a pH of 7.0 or 7.6, containing (in mM): 144.25 NaCl (pH 7.0)/108.48 NaCl (pH 7.6), 2.5 KCl, 1.25 NaH2PO4, and 12 NaHCO3 (pH 7.0)/47.77 NaHCO3 (pH 7.6), bubbled with carbogen. After calibration, electrodes were positioned in layer II/III at 40–60 µm below the slice surface and [K+]o, [Na+]o, or pHo were recorded. Electrodes were calibrated again in the experimental bath directly after each experiment. Data were processed in “OriginPro 2021” and “MS Excel” .Calibration of KM), patch-clamp recordings were performed at an upright microscope equipped with infrared differential interference contrast  and an infrared video camera  using an EPC10 amplifier and “PatchMaster” software . Patch pipettes were pulled from borosilicate glass capillaries (GB150(F)-8P, Science Products, Hofheim am Taunus, Germany) at a vertical puller  and had a resistance of 3.5–4.5 MΩ.To measure astrocytic membrane potential  of the recorded cell i upon transient energy depletion, simulating the situation in the ischemic penumbra. Energy failure was induced by transient inhibition of glycolysis and oxidative phosphorylation  as described earlier i in astrocytes in situ, we employed a combined empirical–theoretical approach. The latter exploits the dominating K+ permeability of the plasma membrane of astrocytes, which, together with a minor permeability for Na+, allows an approximation of their membrane potential (EM) by a simplified GHK equation [z]o/i are respective ion concentrations outside and inside of the cell, and α is the relative membrane permeability for Na+ versus K+ (PNa/PK). Rearranging Equation (1) then enables the calculation of [K+]i:Owing to the lack of suitable techniques for quantitative experimental determination of i using Equation (2), we determined all other parameters  experimentally in astrocytes in acutely isolated tissue slices, thereby going beyond former studies using this approach .+-sensitive fluorescent dye SBFI was employed to measure [Na+]i [+]i of 12.1 mM [+]i by 24.4 ± 7.2 mM   . In the extracellular space, a baseline [Na+]o of 157.4 ± 1.5 mM was determined using ion-sensitive microelectrodes [+]o decreased by 1.9 ± 1.0 mM  within 158 ± 24 s and then fully recovered within about 3 min upon washout of the drugs  (Wide field imaging with the Nae [Na+]i . Recent  12.1 mM . Perfusifter SC) A. Peak o, revealing a baseline of 2.7 ± 0.3 mM in layer II/III of acute tissue slices  (1–A3). The maximal [K+]o increase was reached within 149 ± 26 s (4). This was followed by a long-lasting undershoot of 0.6 ± 0.9 mM, after which [K+]o slowly approached the initial baseline  (2).Ke slices . Inducti12/12/9) A1–A3. Th9 ± 26 s A4. This fter SC) A2.Na/PK), the EM of astrocytes was recorded in the whole-cell patch-clamp mode. In this configuration, astrocytic [Na+]i and [K+]i were determined by their respective concentrations in the pipette saline (see methods). α was calculated, rewriting Equation (1) as follows and inserting the experimentally determined [Na+]o and [K+]o :For the determination of α . Upon perfusion with metabolic inhibitors for 2 min, astrocytes transiently depolarized by 15.5 ± 3.8 mV  (1–B3). The peak depolarization was reached after 201 ± 65 s (4), where after EM showed an undershoot by 4.8 ± 3.2 mV. Subsequently, EM fully recovered to baseline within about 11 min after reperfusion with standard saline   B1–B3. Th1 ± 65 s B4, where+]i, the individual traces of the experimentally determined parameters  were averaged . In the following, [K+]i was calculated for each time point using Equation (2). This revealed an average baseline of astrocytic [K+]i of 146.1 mM  and the experimentally determined maximum Na+ load of astrocytes (24 mM). This apparent cation imbalance requires additional transport of charged molecules across the membrane to maintain electroneutrality. One possibility is the passage of Cl−, but recent work indicated that [Cl−]i of neocortical astrocytes does not undergo significant changes in response to brief chemical ischemia  is intimately linked to pH via the activity of carbonic anhydrases  was solved for [HCO3−]o to enable calculation of changes in [HCO3−]o in response to chemical ischemia as shown in The Henderson–Hasselbalch equation, which describes the relation between pH and [HCO3−]i  to calculate [HCO3−]i D as repo3− to the cation–anion balance during transient chemical ischemia, we first subtracted the averaged traces of astrocytic [Na+]i and [K+]i. This uncovered a biphasic anion gap, predicting an additional loss of anions during the initial decline in [K+]i, which turned into an anion gain during the overshoot in [K+]i , however, even aggravated the remaining anion gap in the second phase of recovery . More recently, genetically encoded K+ nanosensors were developed, which owing to their low apparent affinities for K+, are more suitable for the determination of extracellular [K+] [+]i in HEK cells [+]i in brain tissue has not been established yet.While it is widely accepted that glial cells maintain a high [K+]i ,50, dire0–170 mM ,27,28,510–170 mM ,53,54,55trocytes . This dylar [K+] ,25. A seEK cells , but its+]i and changes thereof in response to energy deprivation in astrocytes in situ, we, therefore, followed earlier approaches combining experimental determination of different cellular parameters with calculation of [K+]i using the GHK equation. This strategy is based on the observation that mature astrocytes express a battery of different K+ channels, including inwardly rectifying Kir4.1, the two-pore domain channels (TWIK-1 and TREK-2) as well as large-conductance calcium-activated K+ channels (BKCa) o and [K+]i i ,58.+]i, provided that all other parameters are known. Here, going beyond earlier reports, we not only measured [K+]o, [Na+]o and EM, but also [Na+]i as well as the relative Na+ permeability in our preparation experimentally, enabling a realistic approximation of [K+]i. We determined a relative membrane permeability for Na+ versus K+ (PNa/PK) of 0.01 in neocortical astrocytes, which is lower than that of astrocytes in the mouse hippocampal CA1 area , we obtained a baseline [K+]i of 146 mM for neocortical astrocytes. This value is somewhat higher than that reported for astrocytes cultured from the mouse cortex (133 mM) [+]o, they were most likely more depolarized, probably yielding a lower [K+]i as compared to our study.The GHK equation evidently also allows for calculation of o in the brain [+]i in response to neuronal stimulation and/or a rise in [K+]o o elevation o ,51,52,59levation ,60,61,62+]o homeostasis is one of the first consequences of anoxic/ischemic conditions and a restriction in cellular energy supply [D of 2.7 mM for ATeam1.03YEMK in both neurons and astrocytes. A linear plot between 1 and 3 mM ATP showed that a 10% change in fluorescence ratio corresponded to a change in [ATP]i by 0.56 mM. Based on this calibration o, an increase in [K+]o, an increase in astrocytic [Na+]i, and a depolarization of astrocytes, confirming earlier observations [+]o and glial EM showing an additional undershoot below baseline and hyperpolarization, respectively i by about 43 mM upon brief inhibition of cellular energy production, most likely as a direct consequence of the decline in cellular ATP and the resulting decrease of NKA activity. Besides channel-mediated loss of K+, activation of astrocytic glutamate transporters might, at least partly, contribute to the decline in astrocytic [K+]i i ,23.+ efflux from astrocytes under ischemic conditions will aggravate the accumulation of extracellular K+ induced by its release from neurons, our simulation also predicts a delayed gain of astrocytic K+ during the late recovery phase. This phenomenon arises from the mismatch in the experimentally determined time courses of the undershoot in [K+]o and the astrocytic hyper- and repolarization, respectively. It again suggests ongoing increased activation of the NKA following metabolic inhibition, probably because of the still-elevated [Na+]i and/or in response to an increase in [Ca2+]i [+ uptake might at least partly be mediated by increased activity of inward NKCC1 [While initial K [Ca2+]i ,58,60,69rd NKCC1 ,36,70.+ by astrocytes reduces [K+]o below its initial baseline. The latter will exert a hyperpolarizing effect on neurons and reduce neuronal excitability [+]o-mediated dampening of network activity and reduction of neuronal ATP consumption, supporting the recovery of neurons from metabolic stress.Notably, the overcompensating uptake of Ktability ,50, whiltability . Astrocy+ movements across the astrocytic membrane predicted by our simulation require the additional flux of ions to preserve electroneutrality i even adds to the delayed predicted gain of K+ in the second phase of recovery. This mismatch in the cellular cation–anion balance must be balanced by an additional net gain of cations, followed by additional cation loss in the second recovery phase and/or an initial net loss of anions, followed by a gain of anions. Besides loading with Na+, astrocytes are subject to a transient increase in [Ca2+]i [+]i (this study) during brief chemical ischemia. While this might reduce the anion gap in the first phase, the slow recovery from both Ca2+ signals and acidification result in its aggravation in the second phase.The Kutrality . While N studies ,67. What [Ca2+]i  and [H+]3− [i and [HCO3−]i, respectively, in response to chemical ischemia, significantly reducing the predicted imbalance in the first phase. The slow recovery from the intracellular acidification and ongoing decrease in [HCO3−]i, however, will again worsen the anion gap in the second recovery phase. While the initial charge imbalance may thus be compensated for through influx of additional cations  and a reduction in [HCO3−]i, these processes will later even increase the predicted anion gap.A mobile anion which will contribute to the cation–anion balance is HCO3− . Our exp− is another candidate for compensation of the predicted delayed charge imbalance. Earlier work has demonstrated rapid channel- and transporter-mediated movement of Cl− across astrocytic membranes [− moreover contributes to astrocyte swelling in ischemic conditions [−]i in response to a chemical ischemia for 2–5 min [− accumulation (NKCC1) and Cl− efflux (KCC’s and glutamate transporters) [−]i was, however, observed upon a 10 min’ exposure to the metabolic inhibitors, showing that a net gain in Cl− is indeed observed upon more severe conditions . On the other hand, earlier reports demonstrating an apparent mismatch between K+ and Cl− movement in astrocytes speculated that in addition to Cl−, the net flux of lactate or other anionic osmolytes such as taurine, glutamate or aspartate might help maintaining the cellular cation–anion balance i. Several factors could have biased our simulation. Among them is the influence of space clamp errors in patch-clamp measurements, a factor especially critical for astrocytes, which not only exhibit a very low input resistance but are also extensively coupled via gap junctions o, thereby exerting a dampening effect on neuronal excitability. Conversely, any net gain of osmolytes by astrocytes will eventually be accompanied by uptake of water, which will promote harmful cell swelling upon prolonged metabolic inhibition [With these considerations in mind, we conclude from our data that the predicted net gain of Khibition ,75,80. Ihibition ,76.


Dear Editor,Microglia‐mediated neuroinflammation plays a crucial role in subarachnoid haemorrhage (SAH).Tmem119 and Cx3cr1 for microglia, others in Method S9), we classified single‐cell transcription into microglia (n = 5824) and other six cell types   and healthy (n = 8160) mouse brain microglia and named it the integrated microglial cluster (IMG‐C) for supplementary analysis  murine model) Figure . We term) Figure . We furt Figures .Spp1, Lpl, Apoe, Ctsb, Lgals1, Lgals3, Fabp5, Mif, Lilrb4a, Cst7 and Vim. Other genes with high expression in SMG‐C5 included Anxa2, Apoc2, CD63, CD72, Ctsc and Ctsz—that have also been found to be upregulated in developing mouse brain microglia, Alzheimer's disease (AD) and lysolecithin (LPC) injury mouse modelsSpp1, Apoe and Lpl,Trem2, Clec7a, Ccl6, or Axl, but SAM has a unique panel of highly expressed genes, such as Spp1, Lgals1, Lgals3, Fabp5, Mif, Lilrb4a and VimSpp1, Lgals, Mif and Fabp5 are related to the enhancement of myelination, microglia regulation and activation.Among microglia in the SAH‐affected brain, three distinct subsets of SMG‐C5 (SAH‐associated microglia‐SAM), SMG‐C6 and SMG‐C7 were observed, whereby the molecular features of SMG‐C5 and SMG‐C6 differed from the previously reported activating microglial statesIl1a, Il1b, Tnf, Ccl4 and Ccl3), chemokines (Cxcl10 and Cxcl2), and other immune signal‐regulating genes  (Figures Il1a, Ccl4 and Ccl3 has been observed in the IRM subpopulation in the MS mouse model.Birc5, Mki67 and Fabp5 in SMG‐C7 and the metabolically active genes Mif and Ms4a6c in SMG‐C5.SMG‐C6 expressed a variety of cytokines ( Figures . Some ofWe analysed the interaction of microglial subsets in the SAH state and found that microglia in SMG‐C5, SMG‐C6 and SMG‐C7 had the most frequent inflammatory pathway connection Figures . The eleIn summary, transcriptional analysis revealed diverse SAH‐specific microglial subgroups and found a functional relationship of elevated signalling pathways through receptor–ligand interactions with microglial subsets. Microglia and other cell crosstalk further substantiates the importance of microglia in driving post‐haemorrhage responses. However, the inclusion of only male mice was a limitation of our research. In addition, the microglial transcriptome at multiple time points after SAH and sham operation is needed in the future. Collectively, our research could be a starting point, provide data and theoretical support for future work and may potentially accelerate the development of microglial inflammation modulators of SAH.Junfan CHEN and Kwok Chu George WONG designed the study. Junfan CHEN carried out most of the experiments, bioinformatics analysis, manuscript writing, revising and figure preparation. Lei SUN, Hao LYU, Huasheng LAI, Zhiyuan ZHENG performed the SAH model establishment and some of the immunostaining. Lei SUN, Sheng GUAN, Yisen ZHANG and Yang WANG assisted in bioinformatics analysis. Yujie LUO, Gang LU and Wai Yee CHAN contributed to the conceptualization of the study. Xinyi CHEN and Zhongqi LI helped with the microglial isolation steps. Junfan CHEN, Ho KO and Kwok Chu George WONG wrote the manuscript with input from all authors.The authors declare no conflict of interests.Supplementary InformationClick here for additional data file.Supplementary InformationClick here for additional data file.Supplementary InformationClick here for additional data file.Supplementary InformationClick here for additional data file.Supplementary InformationClick here for additional data file.


Background: Ischemic stroke is the most common stroke incident. Sphingosine-1-phosphate (S1P) receptor 3 (S1PR3) is a member of the downstream G protein-coupled receptor family of S1P. The effect of S1PR3 on ischemic stroke remains elusive.Methods: We downloaded two middle cerebral artery occlusion (MCAO) microarray datasets from the Gene Expression Omnibus (GEO) database and screened differentially expressed genes (DEGs). Then, we performed a weighted gene coexpression network analysis (WGCNA) and identified the core module genes related to ischemic stroke. We constructed a protein–protein interaction (PPI) network for the core genes in which DEGs and WGCNA intersected. Finally, we discovered that S1PR3 was involved as the main member of the red proteome. Then, we explored the mechanism of S1PR3 in the mouse tMCAO model. The S1PR3-specific inhibitor CAY10444 was injected into the abdominal cavity of mice after cerebral ischemia/reperfusion (I/R) injury, and changes in the expression of blood–brain barrier-related molecules were measured using PCR, western blotting, and immunofluorescence staining.Results: Both GEO datasets showed that S1PR3 was upregulated during cerebral I/R in mice. WGCNA revealed that the light yellow module had the strongest correlation with the occurrence of IS. We determined the overlap with DEGs, identified 146 core genes that are potentially related to IS, and constructed a PPI network. Finally, S1PR3 was found to be the main member of the red proteome. In the mouse cerebral I/R model, S1PR3 expression increased 24 h after ischemia. After the administration of CAY10444, brain edema and neurological deficits in mice were ameliorated. CAY10444 rescued the decreased expression of the tight junction (TJ) proteins zonula occludens 1 (ZO1) and occludin after ischemia induced by transient MCAO (tMCAO) and reduced the increase in aquaporin 4 (AQP4) levels after tMCAO, preserving the integrity of the BBB. Finally, we found that S1PR3 is involved in regulating the mitogen-activated protein kinase (MAPK) and (phosphatidylinositol-3 kinase/serine-threonine kinase) PI3K-Akt signaling pathways.Conclusion: S1PR3 participates in the regulation of blood–brain barrier damage after cerebral I/R. S1PR3 is expected to be an indicator and predictor of cerebral ischemia, and drugs targeting S1PR3 may also provide new ideas for clinical medications. Acute ischemic stroke (IS) accounts for >80% of all strokes . The gloWe focused our attention on IS, downloaded two middle cerebral artery occlusion (MCAO) microarray datasets from the GEO database, screened them for differentially expressed genes (DEGs), performed weighted gene coexpression network analysis (WGCNA) analysis, and identified core genes related to IS. We used the core genes at the intersections of modular genes, DEGs, and WGCNA to construct the protein–protein interaction (PPI) network and finally determined the expression of the core gene sphingosine-1-phosphate (S1P) receptor 3 (S1PR3). S1PR3 is a member of the family of G protein-coupled receptors downstream of S1P. The metabolites of sphingolipids include ceramide, sphingomyelin, and S1P. As the active product of sphingosine, S1P is a natural lipid with biological activity. S1P is mainly produced from sphingomyelin on the cell membranes of red blood cells, platelets, and endothelial cells through a reaction catalyzed by various enzymes . After SThe central nervous system is rich in S1P, and the content of SIP in the brain is higher than that in other organs, suggesting that it may be an important source of S1P in the blood. The destruction of the BBB caused by cerebral ischemia initiates the development of brain edema and the progression of brain damage . In thishttp://www.ncbi.nlm.nih.gov/geo) as a screening set. The datasets were collected from the brain tissues of model mice and normal mice subjected to MCAO. Finally, three normal samples from GSE30655 and four normal samples from GSE35338 were included, including seven MCAO samples in GSE30655 and five MCAO samples in GSE35338. The GSE22255 dataset based on the GPL570 platform was downloaded for verification. This dataset was used for gene expression profiling in peripheral blood mononuclear cells (PBMCs) from 20 patients with IS and 20 healthy people. In addition, in WGCNA (Two microarray datasets (GSE30655 and GSE35338) based on the GPL1261 platform were downloaded from the Gene Expression Omnibus (GEO) database , and an average link hierarchical clustering analysis was performed. The related modules were classified according to the TOM, and the number of genes in each module was not less than 50. The shear height of the gene modules in this study was 0.7, and similar modules were merged. In addition, the gene significance (GS) and module member (MM) in each module were calculated to draw scatter plots. Finally, the Pearson method was used to calculate the correlation between the combined module and MCAO.First, we screened DEGs in the screening set and used the limma package to determine the DEGs between MCAO samples and normal samples. We selected |log2 fold change (FC)|>1 and adjusted p-value < 0.05 and q-value > 0.05.Gene Ontology (GO) enrichment analysis is a commonly used bioinformatics method for searching for comprehensive information in large-scale genetic data, including biological processes (BPs), molecular functions (MFs), and cellular components (CCs). . In addihttp://metascape.org) was used to construct the PPI network of these specific genes, and the MCODE method was used to identify the closely related proteome in the PPI network. Cytoscape software was used to visualize the network.The Metascape database . After 24 h, the heart was perfused with normal saline; the brain tissue was taken out quickly and weighed; and 50% trichloroacetic acid was added, homogenized by a homogenizer, at 4°C, and centrifuged at 3000 rpm/min for 20 min. The absorbance of the supernatant was measured by a spectrophotometer at 620 nm. Draw the standard curve according to the instructions, dilute Evans blue with normal saline, take appropriate amount to add trichloroacetic acid solution, and then dilute, spectrophotometer to measure the wavelength, and finally calculate the content of Evans blue according to the absorbance.Brain sections were fixed with 4% paraformaldehyde. After permeabilization and blocking, the samples were incubated at 4°C overnight with primary antibodies specifically raised against the following proteins: CD31 , zonula occludens 1 (ZO1) , occludin , aquaporin 4 (AQP4) , and matrix metalloproteinase-9 (MMP9) . Subsequently, the samples were incubated with the appropriate fluorophore-conjugated secondary antibodies  for 1 h at room temperature in the dark. DAPI  was used to stain cell nuclei. Images were captured using a fluorescence microscope . We used ImageJ to analyze the immunofluorescence area, and the fluorescence images were imported into the software. Ensure the image is eight bits, and convert to grayscale to best visualize all positive staining. Then, adjust brightness and contrast, go to “Image” and select “Adjust” and “Threshold.” Adjust the upper and lower pulleys, select the positive signal area, and click “Analyze” and “Measure” to obtain statistical results. The immunofluorescence image area we selected is the peri-ischemic regions of the ischemic hemisphere. The number of mice in each group was four, and each group had at least four independent sections. The experiment was double-blind, and the group members were not the same as the experimenter who read images and performed statistics.TRIzol reagent  was used for PCR, and the fasting one-step method was used to remove genomic DNA. The cDNA first-strand synthesis premixes the reagent to synthesize first-strand cDNAs . Finally, quantitative PCR was performed using a PCR machine  with the SYBR green kit , and the relative gene expression level was normalized to GAPDH. The primer sequences were as follows:Occludin: F: TTGAAAGTCCACCTCCACCTCCTTACAGA, R: CCGGATAAAAAGAGTACGCTGG;ZO1: F: GCCGCTAAGAGCACAGCAA, R: GCCCTCCTTTTAACACATCAGA;MMP9: F: GCAGAGGCATACTTGTACCG, R: TGATGTTATGATGGTCCCACTTG; AQP4: F: CTTTCTGGAAGGCAGTCTCAG, R: CCACACCGAGCAAAACAAAGAT; and GAPDH: F: GGTGTGAACCATGACAAGTATGA, R: GAGTCCTTCCACGATACCAAAG.Brain tissue from the right hemisphere was obtained, and proteins were extracted on the first day after ischemia and reperfusion. A BCA protein concentration determination kit  was used to determine the protein concentration. In addition, 30 µg of protein from each group were loaded onto an 8% or 10% SDS-PAGE gel. After electrophoresis, the brain proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% skim milk at room temperature for 1 h, and then incubated with the primary antibody overnight in a 4°C refrigerator. After incubation with goat anti-mouse and anti-rabbit  secondary antibodies for 45 min at room temperature, membranes were washed with TBST and then incubated with enhanced chemiluminescence (ECL) reagent  for detection. Primary antibodies included anti-S1PR3 , anti-AQP4 , anti-MMP9  anti-ZO1 , anti-Occludin , anti-ERK1/2 , anti-phospho-ERK1/2 , anti-AKT1/2/3 , anti-phospho-AKT1/2/3 , anti-p38MAPK , and anti-phospho-p38MAPK . β-Tubulin  was used for internal comparison. ImageJ software was used to quantitatively analyze the grey values of all protein bands.p < 0.05 was regarded as statistically significant.GraphPad Prism software 8.0 was used for the statistical analysis. The data are presented as the means ± SEM. Differences between groups were analyzed using one-way ANOVA followed by the Tukey post hoc test. First, we identified 405 DEGs in GSE30655. The heatmap shows the top 10 differentially expressed genes based on logFC . The volp-value < 0.05, which were included as the input matrix. The clustering of each sample was better  24 h after tMCAO . We also injected mice with a specific inhibitor, CAY-10444, into the peritoneum during reperfusion. The brain water content in mice was measured to evaluate cerebral edema induced by cerebral I/R injury. Compared with the sham group, the brain water content was significantly increased after tMCAO (p < 0.001) (p < 0.001). Compared with the V+24 h tMCAO group, the brains of mice treated with CAY-10444 also showed less water accumulation in the cells (p < 0.001). The BBB infiltration was assessed by the detection of Evans blue exudation. Compared with the sham group, the exudation of EB was significantly increased after tMCAO (p < 0.001) but decreased after CAY10444 (p < 0.001)  . The bra< 0.001) . It suggp < 0.01), indicating that the integrity of the BBB was damaged after IS. However, the administration of S1PR3 inhibitors rescued the expression of these TJ proteins after tMCAO (p < 0.05) . At the same time, S1PR3 inhibition upregulated the level of the ZO1 and occludin transcript (p < 0.05)  (p < 0.05) (The BBB is mainly composed of tight junctions (TJs) formed by protein–protein interactions between endothelial cells, such as ZO1 and occludin, which play a key role in maintaining the integrity of the BBB. Therefore, we detected the expression of these TJ proteins using Western blot and immunofluorescence analyses. On the first day after tMCAO, the expression of ZO1 and occludin decreased significantly ( < 0.05) . Similar < 0.05) . The resctively) . However < 0.05) , which w < 0.05) . Based op < 0.01 and p < 0.05, respectively) , CAY-10444 administration decreased the expression of AQP4 (p < 0.05). At the same time, the inhibition of S1PR3 downregulated the level of the AQP4 transcript (p < 0.05) . At the transcript level, the 24 h tMCAO group exhibited increased expression of the MMP9 mRNA compared with the sham group (p < 0.001), but inhibition of S1PR3 did not affect the level of the MMP9 transcript (p = 0.976) (p < 0.05) (p < 0.05) (p < 0.05) (p < 0.01)  . Then, t of AQP4 . Similar < 0.05) . After t= 0.976) . Under p < 0.05) . After C < 0.05) . Immunof < 0.05) . Compare < 0.05) , althoug < 0.01) .p < 0.001 and p < 0.05, respectively). When the S1PR3 activity was inhibited by CAY-10444, the increase in the phosphorylation of ERK1/2 and p38 MAPKs was reduced  (p < 0.001), and this decrease was reversed by CAY-10444 administration (p < 0.01)  . In cont < 0.01) . Accordi < 0.01) . AQP4 ex < 0.01) . TherefoIS is caused by the sudden interruption of arterial blood flow to brain tissue; then, TJs between cerebral microvascular endothelial cells are destroyed, and the expression of the trans-BBB-specific transporter is restricted . The perin vitro and in vivo , of which MMP9 is mainly localized in astrocytes . PreviouIn addition, we verified the results in the GSE22255 peripheral blood sequencing collection and found that S1PR3 was significantly upregulated in patients with IS and was a good IS indicator; therefore, S1PR3 can be used as a marker molecule for predicting cerebral ischemia.This article identified that the upregulated S1PR3 was a core gene related to IS by screening differentially expressed genes in the GEO dataset, performing WGCNA, and constructing the PPI network. Then, we performed experimental verification of our results and finally found that S1PR3 participates in the destruction of the integrity of the BBB during cerebral ischemia and reperfusion injury and participates in the regulation of PI3K/Akt, p38MAPK, and ERK1/2 pathways. S1PR3 also represents a potential predictor of cerebral infarction. However, more experiments are needed to verify the role of S1PR3 in cerebral ischemia. These results will help clarify the potential mechanism of BBB destruction after cerebral ischemia at the molecular level, and CAY-10444, a specific inhibitor of S1PR3, is expected to become a clinical drug for cerebral infarction, providing guidance for the pathogenesis, diagnosis, and treatment of cerebral infarction.


To overcome the inherent shortcomings of the CNTs' solubility and dispersion in both organic phases and in the sol–gel solution, the outer surface of the CNTs was initially functionalized with carboxylic acid groups and then reacted with both aramid oligomers and 3-aminopropyl triethoxysilane (APTES). The obtained sorbent was characterized by FT-IR, scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). Using sol–gel chemistry, the functionalized CNTs were coated onto SPME fibers and used in conjunction with GC-MS for the analysis of polycyclic aromatic hydrocarbons (PAHs) in water and soil samples. Excellent repeatability (run-to-run RSD% ∼ 8) and reproducibility (fiber-to-fiber RSD% ∼ 6) were achieved in addition to low LODs (0.10–0.30 ng mL−1) and noticeable recovery%. The present method of sorbent preparation led to enhanced thermal and chemical stabilities, a long sorbent lifetime and good affinity towards PAHs. Moreover, the present sorbent enhanced the extraction capability by more than 30% compared to that of commercially available PDMS counterparts.This work describes the preparation of an analytical microextraction sorbent using a simple and versatile sol–gel hybrid composite,  i.e., aramid oligomers wrapping multi-walled carbon nanotubes (CNTs) covalently bonded to a porous silica network.This work describes the preparation of an analytical microextraction sorbent using a simple and versatile sol–gel hybrid composite,  PAHs are now considered priority pollutants by the United States Environmental Protection Agency (US-EPA).4–6 Accordingly, different extraction techniques, such as solid-phase extraction,7–9 SPME,10,11 dispersive micro-solid phase extraction,12–14 liquid–liquid extraction,15 and dispersive liquid–liquid micro-extraction combined with micro-solid-phase extraction, have been applied to isolate PAHs from water samples.16 Owing to the presence of PAHs in trace quantity in the matrix, an effective enrichment method should provide good accuracy, analytical microextraction performance, stability, selectivity, and loading capacity.17–22 However, some of these methods are time-consuming, lack automation or portability, and some consume large amounts of solvents. Therefore, innovations in solid-phase microextraction, which is a simple, portable, solvent-less and non-exhaustive method, are continuously needed to find new sorbents that are capable of providing enhanced analytical performance.Polycyclic aromatic hydrocarbons (PAHs) are considered to be toxic and carcinogenic in nature; however, due to increased human activity, the level of light PAH pollution is dramatically increasing with time.23,24 However, CNTs suffer from self-aggregation and stacking phenomena via π–π and van der Waals interactions, resulting in limited solubility in organic solvents. Therefore, various efforts have been made to functionalize the outer surface of CNTs to enhance and improve their solubility and dispersion in different solvents. For example, Sarafraz-Yazdi's group investigated the functionalization of CNTs with polyethylene glycol (PEG) polymeric tails to enhance their dispersion and compatibility in sol–gel matrices.25 Additionally, many modified CNTs with surfactants,26 amino acids,27 cyclodextrins,28,29 polythiophene,30 and polyaniline–polypyrrole31 have been prepared and their utility as microextraction sorbents has been evaluated. Moreover, nitrogen-doped CNTs with excellent analytical performance for the extraction of polychlorinated biphenyls (PCBs) have been described.32 In addition, other approaches have been utilized such as the electrodeposition of the layer-by-layer CNTs-ZIF-polyaniline sorbent for the extraction of PAHs, the use of oxidized CNTs as solid-phase extraction (SPE) sorbents for the extraction of polar analytes,33 and pristine CNTs for dispersive SPE,34,35 magnetic DSPE and molecularly imprinted polymers.36 The surface modification and functionalization of CNTs have been effectively used for the covalent linkage of CNTs with the microextraction media.25,37,38 Ahmad et al. proposed a convenient and robust method for the modification of acid-functionalized CNTs with aramid (Ar) oligomers via the aramid linkage (–ph–CO–NH–ph–).39 Ar-Chains on the CNT outer surface lead to better compatibility, miscibility, and dispersion of Ar-CNTs in organic solutions.Single or multi-walled carbon nanotubes (CNTs) have been utilized as extractive sorbents due to their chemical stability, high surface area, and high surface-to-volume ratio.40,41 has offered a reasonable solution for the inherited drawbacks of conventional coating methods for SPME such as poor chemical and thermal stabilities. The sol–gel coating method has been widely investigated42–48 since it was introduced by Malik via hydrolytic and non-hydrolytic routes.49,50 Herein, an efficient, simple, and robust route has been developed for the preparation of a homogeneous organic–inorganic sorbent comprising aramid-wrapped carbon nanotubes covalently bonded into a sol–gel network, and a sol–gel binder  was used to play a dual role: (a) binding with the functional groups on the surface of the CNTs to create sol–gel “active” CNTs and (b) linking the aramid oligomers and the functionalized CNTs. This sol solution was then used for the preparation of the SPME sorbent.Sol–gel chemistry1,4- and 1,3-phenylene diamines and terephthaloyl chloride (TPC) were both of analytical grade (99% pure). Oxidized CNTs  with a purity of 95% were purchased from Nano Armor USA (4% oxidation). Pyrene, naphthalene, fluoranthene, acenaphthylene, phenanthrene, hexane, acetone, sodium hydroxide, 3-aminopropyl triethoxysilane (APTES), tetrahydrofuran, and anhydrous dimethylacetamide (DMAC) were purchased from Sigma Aldrich. Hydrochloric acid was obtained from J. T. Baker USA. Silica gel beads were obtained from Merck, while fused silica untreated fiber was purchased from Supelco.−1 from ambient temperature to 800 °C under a nitrogen atmosphere. FT-IR spectroscopy on the samples was performed on a Jasco spectrometer  within the frequency range of 400–4000 cm−1 using the pressed KBr disk technique. Chromatographic experiments were performed on a Shimadzu GC-2010  equipped with a mass spectrometry detector (GCMS-QP2010 Series). A Forte  GC capillary column (25 m × 0.25 mm ID and 0.25 μm thickness) was used for the separation. The total flow rate was maintained at 52.2 mL min−1. Helium was used as a carrier gas with a 1 mL min−1 flow rate. The injection port was set at 300 °C. The column temperature program: the initial temperature was 60 °C for one min, the hold-time temperature was then raised to 180 °C at a rate of 30 °C min−1, and afterwards at 30 °C min−1 till 280 °C and held for 5 min. Commercial SPME fiber  was generously provided by KU NUERS.The morphology of the synthesized material was investigated using scanning electron microscopy (SEM). The samples were mounted on aluminum stubs, gold-coated using a Lecia ACE 200 Sputter coater, and then observed at 20 kV using a JCM-5700 SEM Carryscope . Thermogravimetric analysis (TGA) was performed using a DTG-60 . Measurements were taken at a heating rate of 10 °C min−1.Soil samples were collected from the gulf coast of Kuwait, crushed, and sieved with 100–200 mesh. The soil samples were used as a blank matrix and stored at 4 °C before use. Seawater samples were collected from Kuwait Gulf Bay using cleaned glass bottles and stored in a refrigerator. Crude seawater samples were filtered under vacuum through a 0.45 mm membrane filter and used later as a blank matrix. The blank soil and seawater samples were spiked with standard solutions of naphthalene, acenaphthylene, phenanthrene, fluoranthene and pyrene at a concentration of 0.3 mg L51 A solution of TPC (0.985 mmol) in DMAC was taken in a 50 mL flask. A homogeneous solution (0.86 mmol) of 1,4- and 1,3-phenylene diamines, taken in a mole ratio of 35 : 65, in DMAC was then added dropwise to the TPC solution at 0 °C with stirring under complete anhydrous conditions  was added to the solution, followed by thorough vortexing. The solution was then heated at 60 °C with continuous stirring for 2 h, followed by the cooling of the resultant solution at room temperature .An aliquot of 3 mL of the aramid oligomer solution was added to 5 mL of the silanized CNTs and the mixture was vortexed thoroughly . The pre−1) was used to prepare the PAH stock solution. PAH solutions (0.01 to 0.1 μg mL−1) were prepared by diluting the PAH stock solution with hexane. Prior to the extraction, the fiber was conditioned at 300 °C in the GC injection port for 1 h. The SPME coated fiber was immersed in 10 mL PAH sample solution for a predetermined extraction time. The coated fiber was then inserted in the GC inlet (at 300 °C for 6 min) for the thermal desorption of the extracted analytes.A standard solution (100 μg mLg. The filtered and centrifuged supernatant was then evaporated under vacuum. The residue was then dissolved with 10 mL deionized water and vortexed and sonicated for 10 min each prior to extraction by immersion (DI-SPME).A spiked soil sample (2 g) was sonicated for 10 min and homogenized by vortexing with 10 mL hexane, which was added to extract the analyte from the soil sample. The mixture was then filtered through a filter membrane through three rounds of filtration. The resulting filtrate was then centrifuged for 10 min at 20k × i.e., the C–O, O–H, and C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretching signals at 1160 cm−1, 3436 cm−1, and 1628 cm−1, respectively, in CNT-COOH52,53  of the aramid oligomer for the formation of the target sorbent, i.e., aramid-wrapped CNTs  confirmed the good sensitivity of the Ar-CNT sorbent toward these carcinogenic pollutants due to the various intermolecular interactions. Excellent recovery% was achieved using the present sorbent in the range of 91 to 103. For a non-exhaustive technique such as SPME, the concept of recovery is related to the extraction process rather than the actual removal of the analyte from the sample matrix since non-exhaustive techniques extract at minimum micrograms or even nanograms of the analyte. For recovery% evaluation, the original sample concentration was compared to the sample concentration revealed by the extraction process, which is described by the following equation:n is the amount extracted, Kfs is the distribution coefficient, Vf is the fiber volume and Co is the original sample concentration.To judge the applicability in real samples, a proof-of-concept experiment was conducted by the extraction of naphthalene, acenaphthylene, phenanthrene, fluoranthene and pyrene from soil and seawater samples. A hybrid, stable, and tunable sol–gel-based sorbent served as an SPME fiber coating. CNTs were efficiently wrapped with aramid oligomers to provide good miscibility and homogeneity in organic phases. Herein, a sol–gel binder was implemented to provide a dual role: covalent functionalization for the outer surface of the CNTs and sol–gel activation. The presence of various intermolecular interactions such as π–π stacking, hydrophilic, and hydrophobic interactions in the prepared sorbent allowed for noticeable affinity toward PAHs in water and soil samples. The prepared SPME sorbent allowed for acceptable levels of the obtained analytical parameters. Excellent sorbent reproducibility (fiber-to-fiber), SPME repeatability (run-to-run), recovery% and detection enhancement were achieved along with a significant lifetime. The method of preparation described in this work can be further utilized for the attachment of different organic moieties into sol–gel organic–inorganic hybrid composites for tunable sorbent design.There are no conflicts to declare.RA-012-D2RA02659G-s001


Porphyridium cruentum. This unicellular marine redalga can secrete and accumulate high-value compounds that can findapplications in a wide variety of industrial fields. 300 ± 67mg/L of exopolysaccharides were obtained from cell culture medium;phycoerythrin was efficiently extracted  andisolated by single chromatography, with a purity grade that allowedthe crystal structure determination at 1.60 Å; a twofold increasein β-carotene yield was obtained from the residual biomass;the final residual biomass was found to be enriched in saturated fattyacids. Thus, for the first time, a complete exploitation ofP. cruentumculture was set up.Here, an unprecedented biorefinery approach has beendesigned torecover high-added value bioproducts starting from the culture of Porphyridiumcruentumis fully exploited to recover high-added-valueproducts.A new concept in biorefinery: From a theoretical point of view, a biorefineryis a combination of multiple integrated processes able to convertthe biomass into a variety of high-added value products, in an economicaland environmentally sustainable approach.4 This is in line with the principles of circular economy, fosteredby international organizations and promoted by European Union . Unfortunately, in the microalgae field, a real biorefinery hasbeen demonstrated only for few strains,5 and most of the present literature is focused on the extractionof one or two classes of molecules, thus suggesting that the processis not economically feasible.Microalgae capture carbon dioxide duringtheir growth to performphotosynthesis. This implies the production of oxygen and the reductionof carbon dioxide emissions.Porphyridium cruentum is a red marinemicroalga, reservoir of potentially high-added value molecules, suchas carotenoids, sulfated exopolysaccharides (EPSs), B-phycoerythrin(PE), and lipids.9 Carotenoids are well-known antioxidants, also able to counteractmany disorders, from type 2 diabetes, to degenerative diseases orcancer.10 Sulfated EPSs have a chemicalstructure which confer them peculiar rheological properties11 and many biological activities,12 such as antimicrobial, anti-inflammatory,13 hypocholesterolemic,14 antiviralactivities,15 and skin protective activity.16 PE, the main protein found inP. cruentum, has a good market value for differentreasons: (i) in biomedical and molecular applications for its naturalfluorescence; (ii) as a natural red-colored protein to be used asa dye for food and cosmetics, and (iii) in the pharmaceutical industrythanks to its antioxidant activity.17 Finally,recent literature exploited the use of saturated fatty acids as antimicrobialagents and as drug delivery systems.19P. cruentum, here we propose a real biorefinery.For the first time, not only the biomass but also the exhausted mediumwas fully exploited to recover: EPSs from the medium and PE, carotenoidsand lipids from the biomass. Molecules were extracted sequentially,starting from the most valuable one, without affecting the activityof the molecules in the residual biomass.Thus,taking advantage of the chemical composition ofAll chemicals, solvents, and reagents, unlessdifferently specified, were from Sigma-Aldrich .Porphyridium cruentum strain was acquired from CultureCollection of Autotrophic Organism . Preculture of P. cruentum was inoculated in Porphyridium medium20 ina 1 L bubble column photobioreactor (working volume 800 mL) in a roomwith constant temperature (25 ± 1 °C) and light ,collected and lyophilized. Total carbohydrates were quantified bythe phenol–sulfuric acid method, according to Geresh et al.,21 with some modifications, as reported in Gallego.4 A reference curve was obtained by using glucose(0.03–1.0 mg/mL).To processa high volume of medium, the latter was concentrated 10 times by lyophilization,prior to addition of pure ethanol (1:2 v/v); EPSs were then recoveredby centrifugation  for 16 h at 80 °C.22 Then, thesample was dried and acetylated with 25 μL of acetic anhydrideand 25 μL of pyridine and kept at 100 °C for 30 min. Themixture was analyzed by gas chromatography–mass spectrometry(GC–MS) by using an Agilent Technologies instrument , equipped with a HP-5MS 30 m, 0.25 mm, 0.25 μm capillarycolumn. The following temperature program was used to analyze acetylatedmethyl glycosides: 140 °C for 3 min, 140 °C → 240°C at 3 °C/min.LyophilizedEPSs (2 mg) were solubilized with 1 mL of HCl/CHP. cruentum mediumwas carried out by performing total combustion, using the FlashSmartElemental Analyzer, according to Álvarez-Gómez and colleagues.23 Elements  were expressed as % withrespect to the sample’s total weight.The determinationof total carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) inEPSs recovered from g for 30 minat room temperature and resuspended at 10 mgD.W./mL inPBS pH 7.4. Cell disruption was done by: (i) maceration; (ii) freezeand thaw; (iii) French Press, and (iv) sonication. For the maceration,the biomass was kept at 4 °C in agitation for 24 h. For the freezeand thaw method, the biomass was frozen (−80 °C) and thenthawed (37 °C) for five cycles. For the French Press, two cycleswere performed at a pressure of 2 kbar. The ultrasound method wasperformed by operating with MS73 tip at 40% amplitude of the instrument(Bandelin Sonoplus HD 3200) for different lengths of time, from 4to 20 min, , on ice. At the end ofeach step, samples were centrifuged at 5000 g at4 °C for 30 min, proteins were recovered in the supernatant,total proteins were determined by BCA Protein Assay Kit (Thermo Scientific)and then SDS-PAGE analysis followed by Coomassie staining was performed.Phycobiliprotein concentration was determined by the Bennet and Bogoradequations:24Fresh biomasswas harvested by centrifugation at 1200The reported wavelengths correspond to the maximum of absorption ofphycoerythrin, phycocyanin, and allophycocyanin, respectively.562nm/Abs280nm.PE purification was performedby comparing three techniques: anion-exchange chromatography, gelfiltration, and ultrafiltration. Anion-exchange chromatography wascarried out by using a Nuvia-Q resin equilibrated with PBS pH 7.4,and elution was performed using 0.25 M NaCl. Gel filtration was performedby using a Sephadex G-75 equilibrated in PBS pH 7.4. Ultrafiltrationwas carried out with a 10 kDa molecular weight cut-off membrane, andthe process was performed at 4 °C. The fractions obtained byanion-exchange and gel filtration, as well as the retentate obtainedby ultrafiltration, were collected and analyzed by SDS-PAGE followedby Coomassie staining and PE purity grade was calculated by measuringthe ratio Abs25 using a drop containing10 mg/mL PE in 0.25 M ammonium sulfate, 25 mM potassium phosphateat pH 5.0, equilibrated with a reservoir containing 0.5 M ammoniumsulfate, and 50 mM potassium phosphate at pH 5.0. Red crystals werevisible after 1 week .PE crystals were grown by the hanging dropvapor diffusion method26 The crystal wastrigonal, space group R3, with unit cell parameters a = b = 186.59 Å, c = 59.19Å, α = β = 90°, and γ = 120°. Fordata collection statistics, see Table S1 (Supporting Information). The structure of PE was solved by molecularreplacement using PHASER,27 and the PEstructure deposited in the Protein Data Bank under the accession 3V58,28 as a starting model. The structure shows thepresence of two (αβ) dimers in the asymmetric unit. Visualinspection and model improvements were carried out using Coot.29 Refinements were carried out using Refmac 5.0.30R factor and R free values were used to optimize the refinement strategy. The finalmodel, which has good geometries and refinement statistics , was depositedin the Protein Data Bank under the accession code 8B4N. Pymol (www.pymol.org) was used to obtainmolecular-graphics figures.The crystals were soaked in a cryoprotectant solutioncontaining 30% (v/v) glycerol in the reservoir solution and cooledat −173 °C. Starting from one crystal, a high-resolutiondata set was collected at the XRD2 beamline at the Elettra synchrotronin Trieste, Italy, at −173 °C. Data were processed andscaled using Autoproc.4HCO3 at pH 8.0 and acetonitrile(ACN), followed by reduction  and alkylation . Thegel pieces were washed with 0.1 M NH4HCO3 ofpH 8.0 and ACN and subjected to the enzymatic hydrolysis by coveringthem with 40 μL sequencing grade modified trypsin  overnight at 37 °C.Peptide mixtures were eluted, vacuum-dried, and resuspended in 2%ACN acidified with 0.1% HCOOH. Tryptic peptide mixtures were analyzedby MALDI-TOF  to reveal the amino acid sequenceof the three phycoerythrin chains.A single PE crystal was solubilizedin water and analyzed by SDS-PAGE. For in-gel hydrolysis, SDS-PAGEbands were excised from the gel lane, destained by consecutive cyclesof 0.1 M NHnH)n+: 904.46 Da des-Arg-Bradykinin, 1296.68 Da Angiotensin I, 1570.67Da Glu-Fibrinopeptide B, 2093.08 Da ACTH (clip 1–17), 2465.19Da ACTH (clip 18–39), 3657.92 Da ACTH (clip 7–38)).Peptides were identified by MS spectra, acquired using a mass (m/z) range of 400–4000 Da. Peptide mass fingerprintingwas performed by MS digest of homologue phycoerythrin sequences. MALDIMS experiments were performed on a 5800 MALDI-TOF-TOF ABSciex equippedwith a nitrogen laser (337 nm) . The instrumentoperated with an accelerating voltage of 20 kV, a grid voltage at66% of the source voltage, and a delay time at 200 ns. Laser powerwas set to 3500 V for the spectra acquisition. Each spectrum representsthe sum of 10,000 laser pulses from randomly chosen positions on thesame target place. The data were reported as monoisotopic masses.Matrix-assisted laser desorption/ionization(MALDI) mass spectrometry (MS) experiments were performed on a 5800MALDI-TOF-TOF ABSciex equipped with a nitrogen laser (337 nm) . Starting from each band, aliquots of peptidemixture (0.5 μL) were mixed  with alphacyano hydroxycinnamicacid (10 mg/mL) in acetonitrile: 55 mM citric acid (70:30) solution.Calibration was done by using a calibration mixture from AB SCIEX and sonicated . The mixture volume was then adjusted to 20 mLand shaken for 24 h at 250 rpm in a dark room at 4 °C. The supernatantwas collected by centrifugation at 12,000g for 10min, dried under nitrogen stream, and then stored at −20 °C.Carotenoid identification was performed by HPLC-DAD-APCI-QTOF-MS/MS,whereas quantization was done by calibration curves obtained by usingcommercial zeaxanthin and β-carotene, according to a methodpreviously described,5 with some modifications.The analysis of the extracts was carried out in an Agilent 1290 UHPLCsystem (Ultrahigh Performance Liquid Chromatography) equipped witha diode-array detector (DAD), coupled to an Agilent 6540 quadrupole-time-of-flightmass spectrometer (q-TOF MS) equipped with an atmospheric pressurechemical ionization (APCI) source, all from Agilent Technologies . Extracts were solubilized in ethanol (5 mg/mL), filteredthrough 0.45 μm nylon filters, and then analyzed under positiveionization mode, using the following parameters: capillary voltage,3.5 kV; drying temperature, 350 °C; vaporizer temperature, 400°C; drying gas flow rate, 8 L/min; nebulizer gas pressure, 40psi; corona current (which sets the discharge amperage for the APCIsource), 4000 nA. The mass spectrometer was operated in MS and tandemMS modes for the structural analysis of all compounds. The MS andAuto MS/MS modes were set to acquire m/z values rangingbetween 50 and 1100 and 50 and 800, respectively, at a scan rate of5 spectra per second.Carotenoidswere extracted from the dry biomass, either raw or after protein extraction.Extractions were performed in ethanol, as reported by Aremu, withsome modifications.32 Nitrogen flux was used to dry, recover, andweigh lipids. Lipid fractionation was performed by solubilizing themin chloroform and using a commercial prepacked column containing astationary phase made of Florisil. Neutral lipids were eluted withchloroform:methanol , fatty acids with 2% acetic acid indiethyl-ether, whereas phospholipids were eluted with 100% methanol.The recovered fatty acids were then characterized by GC–MS,as previously reported.33Lipids wereextracted on the dried biomass. Both the raw and the two residualbiomasses  were dried at 60 °C for24 h. Lipids were obtained as reported by Bligh and Dyer.Results are reported as mean ofresults obtained after three independent experiments (mean ±SD) and compared by one-way analysis of variance according to Bonferroni’smethod (post hoc) using Graphpad Prism for Windows, version 6.01.P.cruentum. At the end of cell growth, medium, generallyregarded as waste, was collected and polysaccharides were isolatedby precipitation using ethanol or 2-propanol. The EPS content wasmeasured by the phenol–sulfuric acid method and no differencebetween the two applied solvents was observed (ethanol yield 0.100± 0.020 and 2-propanol 0.116 ± 0.020 mg/mL); thus ethanolwas selected because of its wide range of applications.34 The EPS yield was found to be 300 ± 67mg/L of culture medium, which corresponds to an EPS yield of 0.53g/gd.w.biomass. The monosaccharide composition was achievedby GC–MS analysis, after derivatization as acetylated methylglycosides (AMGs). The GC–MS chromatogram disclosed the presenceof mainly xylose (Xyl), galactose , and glucose (Glc). Finally,traces of rhamnose (Rha), glucuronic acid (GlcA), and glucosamine(GlcN) were detected , whereas PE, phycocyanin (PC), and allophycocyanin (APC)concentrations were obtained using the Bennet and Bogorad equationsand UVA light exposure, taking advantage of chromophores present inPE .The analyses revealed two major bands, whose molecular weights correspondedto α and β subunits (17 kDa) and γ subunit (30 kDa)of PE, in each lane. Results suggested ultrasound as the most promisingmethod. As biomass storage is a crucial step in industrial-scale processes,also from a logistically point of view,37 extractions were performed by ultrasound on either fresh or frozenbiomass (stored at −80 °C) for different lengths of time(from 4 to 20 min). At the end of each extraction, the disrupted biomasswas analyzed by SDS-PAGE . Supernatants obtained after 20 min extractionseemed to be the best choice for fresh biomass, as 4 min extractionallowed to recover a PE content of 1.8% .Only the size-exclusion chromatography allowed obtaining pure PE;in particular, this purification step allowed to recover about 80%of PE and to reach a purity grade of 4.0. It is known that a puritygrade ≥4.0 indicates a protein to be used in analytical grade.38 According to the overall results, this approachallowed obtaining high-purity grade PE by only one step extractionand purification. To the best of our knowledge, this is the firsttime that PE was extracted and purified with such a purity grade byusing only a single purification step.42 To determine the protein identityand purity, PE was crystallized, and its X-ray structure was determinedat 1.60 Å resolution. The X-ray structure is constituted by 5990atoms, including five phycoerythrobilin (PEB) chromophores for eachαβ dimer, one methylated Asn in position β72 3 hexamer deviation of 0.146 Å, and the 177 CA atoms of β subunithave an r.m.s. deviation of 0.142 Å. Each αβ dimerhas five PEBs in position α82, α139, β61, β82,and β158. The first PEB, which adopts two alternate conformationsin our structure, is covalently attached to the side chain of Cys82of the α subunit by ring A. The second PEB is bound to Cys139of the same subunit. The other three PEBs are found in the βsubunit. They are bound to the side chains of Cys61, Cys82, and Cys158.The stereochemistry of the chromophores and their interaction withprotein residues are basically identical to that observed in the startingmodel28 and previously described. An exampleof the well-defined electron density maps of the chromophores is reportedin Figure S3A, Supporting Information) showed apeculiar shoulder at 498 nm, ascribed to the phycourobilin chromophoreof the γ subunit. SDS-PAGE analysis  showed the presence of two molecularspecies, whose molecular mass was compatible with α and βsubunits (double bands at about 17 kDa) and with the γ subunit(30 kDa). In situ digestion was performed on the bands and the peptidemixtures were analyzed by MALDI-TOF; peptide mass fingerprinting wasperformed by MS digest of homologue phycoerythrin sequences. The assignmentof each mass spectrometry signal allowed highlighting the peptidesequence along the entire protein sequence. As shown in 45 The heptameric structures of PE,with the two different γ subunits, extracted from PDB code 6KGXare reported in PE purification was performed by comparing three techniques: anion-exchangechromatography, gel filtration, and ultrafiltration. The results ofthe different techniques are reported in the Supporting Information and on the raw one,used as benchmark (as reported in the 32 followed by a solid phaseextraction (SPE). As shown in 19According tothe strategy described in P. cruentum culture.In particular, (i) P. cruentum cultureis completely employed to recover intra- and extracellular class ofhigh-added-value molecules; (ii) the yield of each extracted classof molecules is similar to those obtained when extractions are performedto recover single class of molecules; and (iii) according to the biorefineryprinciples, all the class of molecules have been recovered startingfrom the one with the highest market value. Simultaneous microalgaeculture valorization and carbon capture can contribute to the sustainableexpansion of the microalgae market.The strategy proposed in


Laboratory evaluation of the TK100 DGT showed linear uptakeof Sr over time (2–24 h) up to very high Sr mass loadings onTK100 membranes (288 μg cm–2) and effectiveperformance in the range of pH (3.9–8.2), ionic strength (0.001–0.1mol L–1), and cation competition (50–160mg L–1 Ca in a synthetic soil solution matrix) ofenvironmental interest. Selective three-step elution of TK100 membranesusing hydrochloric acid allowed us to obtain purified Sr and Pb fractionswith adequate (≥75%) recovery and quantitative (≥96%)matrix reduction. Neither DGT-based sampling itself nor selectiveelution or mass loading effects caused significant isotopic fractionation.Application of TK100 DGT in natural soils and comparison with conventionalapproaches of bioavailability assessment demonstrated the method’sunique capability to obtain information on Sr and Pb resupply dynamicsand isotopic variations with low combined uncertainty within a singlesampling step.A method using diffusivegradients in thin films (DGT) for theaccurate quantification of trace-level (μg L As a result,laborious sample purification strategies are required. The diffusivegradients in thin films (DGT) technique23 provides an alternative sampling strategy, offering the potentialto assess Sr and Pb concentrations and isotope ratios in labile, bioavailablefractions in soils accurately and cost-effectively. DGT is an infinite-sinktechnique using hydrogel layers containing binding agents  with high selectivityfor the target analyte(s). Applied in soil, DGT samples kineticallylabile analyte fractions, consisting of free ions, complexes withinorganic and organic ligands, and exchangeable solid phase pools,15 which usually comprise the bioavailable fraction. Consequently,DGT has been shown to be a better predictor for nutrient and contaminantuptake by plants under diffusion-limited conditions while performingequally well to chemical extractants in nondiffusion-limited cases.15 Moreover, the unique capability of DGT for analytepreconcentration and in situ matrix separation maysubstantially facilitate sample preparation for multicollector (MC)ICP-MS, as recently shown for DGT-based sulfur stable isotope measurementsin labile soil sulfate.16In the past, numerous methods have been proposed to assessthebioavailable fractions of Sr and Pb in soil. Most commonly, soil solutionsampling or equilibrium-type chemical soil extractions using nonbufferedsalt solutions  as the binding agent. The study showed that DGT-measuredSr concentrations agreed well with those in natural freshwater. However,lacking selectivity for Sr, the binding layer was quickly saturatedby codissolved cations, limiting its application in DGT to short deploymenttimes (≤20 h) and soft water environments with a very low ionicbackground (≤0.001 mol L–1). In the secondstudy, Garmo et al.18 tested Chelex DGTfor the measurement of Sr among 54 other elements under laboratoryconditions. While this technique works well for divalent transitionmetals and can also be used for isotope ratio measurements of Pb inaquatic systems,19 its performance forsampling Sr is affected by the low binding affinity of the Chelexresin for alkaline earth metals, causing pH-dependent H+ competition effects.18Yet, theanalysis of Sr by DGT represents unique challenges, asevidenced by the lack of an adequate DGT method for sampling Sr. Untilnow, only two research groups have trialed to assess Sr by DGT quantitatively.The first study on Sr uptake from aqueous solutions by DGT was reportedby Chang et al.,t-butylcyclohexano-18-crown-6(crown-ether) functionality (TK100). The HDEHP binds Sr2+ on an organic stationary phase via a H+ ion-exchange mechanism, thereby facilitating complexation of Srby the crown-ether. Surman et al.20 investigatedTK100 in the resin form for batch- and column-type separations of 90Sr from interfering radionuclides and confirmed that it selectivelyadsorbed Sr from natural water samples with quantitative recovery underenvironmentally relevant conditions . Additionally, they demonstratedthat TK100 shows a particularly high affinity for Pb, indicating thepossibility for adsorptive cosampling of both Sr and Pb from the samesample. Therefore, we evaluated TK100 as a selective binding phasefor the simultaneous measurement of labile Sr and Pb concentrationsunder natural competitive conditions by DGT for the first time. Moreover,we investigated if TK100 DGT allows for direct matrix separation withoutsignificant isotopic fractionation during solute sampling to enableaccurate determination of Sr and Pb isotope ratios by MC ICP-MS. Thenew method was comprehensively evaluated under laboratory conditionsand tested on different types of natural soil samples.In thisstudy, we take advantage of the selective adsorption ofSr by a new binding phase with combined di(2-ethyl-hexyl)phosphoricacid (HDEHP) and 4,4′(5′)-bis-Supporting Information. To comply withnotation conventions used in the DGT literature, the symbol c is used throughout this work for both molar and mass concentrationsin solution instead of the designated symbols c andγ recommended by the International Union of Pure and AppliedChemistry (IUPAC).Details on laboratory procedures and materials areprovided inthe 21 In-house quality control standards (preparedby mixing certified multi- and single-element standards) reflectingsample compositions were analyzed every 6–12 samples for qualityassurance. Analytical recoveries were typically within ±5% ofthe target value. Isotope ratio measurements were accomplished usingNu Sapphire (SP017) and Nu Plasma HR (NP048) sector-field MC ICP-MSinstruments . Sample and standard solutionswere diluted to obtain Sr and Pb mass concentrations of 20–75and 2–15 μg L–1, respectively. Standard-samplebracketing (SBB) was applied using solutions of SRM 987  for Sr and SRM 981 for Pb  as the isotopic reference.21 Sample and standard solutions of Sr were spiked with Zr to enableinternal interelemental correction of the instrumental isotopic fractionation(IIF). Pb isotope ratios were corrected for IIF by SSB. Outlier removal,blank correction, and correction of residual Rb (in Sr measurementsolutions only) were accomplished as reported previously.22 Details of instrumental setups and parametersof the MC ICP-MS analyses of Sr and Pb for different experiments areprovided in Table S1. All samples weremeasured in diluted HCl (w = 2%).Elementalmass concentrationsin sample solutions were measured using quadrupole single-collectorICP-MS . External calibration and internalnormalization using indium were applied as reported previously.Figure S1). To overcomethis problem, a poly(tetrafluoroethylene) (PTFE) membrane , which had not been used for DGT binding layersbefore, was used as the reversed-phase support for the TK100 extractantsystem. The PTFE membrane was impregnated with TK100 at a mass fractionof 37 ± 3% . The TK100 membranes werecut to discs (Adisc = 4.52 cm2) using a stainless-steel cutter, precleaned in HNO3 (c = 0.5 mol L–1) for 6 h under constantshaking, thoroughly rinsed with water until the pH of the storagewater was ∼5, and then stored in water at 6 °C in thedark until use.We aimed to develop a TK100-containingDGT binding layer using polyacrylamide- or polyurethane-based hydrogelmatrices typically used in DGT applications. Despite our efforts toimprove the gel formulation, initial tests showed that the Sr bindingfunctionality of the TK100 resin  is compromised when incorporated into thesehydrogel matrices (23 and a poly(ether sulfone) (PES) filter membrane  on top of each other intoDGT moldings. APA gels and PES membranes were pre-equilibrated inNaNO3 (c = 0.01 mol L–1) for ≥24 h to avoid charge-induced disequilibrium artifacts.17 Assembled DGT devices were stored at room temperaturein plastic zip-lock bags containing ∼5 mL of NaNO3 (c = 0.01 mol L–1) until use.TK100 DGTdevices were assembledby stacking a TK100 membrane disc, an agarose cross-linked polyacrylamide(APA) diffusive gel (0.8 mm thickness),m,bound by the TK100 membrane disc in the DGT device according to i.e., solution or soil) interface, cDGT, using ce (μgL–1) is the analyte mass concentration in the eluate, Vbl (mL) is the TK100 membrane disc volume, Ve (mL) is the eluent volume, fe is the analyte elution factor, Δg (cm) is the diffusive-layer thickness, D (cm2 s–1) is the diffusion coefficient of theanalyte in the diffusive layer, Ap (cm2) is the sampling window area of the DGT device, and t (s) is the deployment time. Sr and Pb isotope ratios insample (spl) solutions are reported relative to an isotopic referencestandard  using the delta (δ in ‰) notationaccording to δ values of the DGT eluate(DGT) and the reference solution (ref), i.e., theimmersion solution (soln) or the NH4NO3 soilextract (NH4NO3), using eq iE is theheavier isotope i of the element E , jE is the lighter isotope j of the element E , and R is the corresponding isotope ratio of the sample or standardafter blank and interference correction. Details on the estimationof combined measurement uncertainties (uc) are provided in the Supporting Information. Significance of differences between two mean values was determinedconsidering their expanded uncertainties .24 Unless stated otherwise,all values in the text are reported as means ±U (k = 2).Thedetermination of the total analyte mass, fu, and elutionrecoveries, fe, of Sr and Pb by TK100membrane discs were evaluatedin mass balance experiments using ICP-MS analysis.17 The discs were deployed in quadruplicates for 24 h in 10mL of well-shaken NaCl solution (c = 0.01 mol L–1) containing Sr and Pb (both c =500 μg L–1) at pH 5.2. The discs were retrievedfrom the immersion solution using pipette tips and tweezers, rinsedtwo times on each side with water using a spray bottle, and placedinto polyethylene (PE) vials. The elution recovery was determinedby batch elution for 24 h in well-shaken 10 mL of HCl (c = 6 mol L–1), which was based on previous workson the TK100 resin, indicating minimal binding of both Sr and Pb underthese conditions.26 Each experiment was repeated at least two times on different daysto assess the uptake and recovery reproducibility.The uptake efficiencies, c = 5000 μg L–1), Rb (c = 500 μg L–1),and Hg (c = 500 μg L–1).To test a potential three-step elution scheme to chromatographicallyseparate Sr and Pb from the potentially cobound matrix interferences,the loaded TK100 discs were retrieved, rinsed, and placed into PEvials as described above. In the first step, elution for 1 h in 10mL of either HNO3 (c = 8 mol L–1) or HCl (c = 0.01 mol L–1) wastested as a matrix wash to remove Ca, Rb, and Hg with minimal lossesof Sr and Pb. In the second step, TK100 discs were placed in 10 mLof HCl (c = 2 mol L–1) for 24 hto elute Sr. In the third step, 10 mL of HCl (c =6 mol L–1) for another 24 h was used to elute morestrongly bound Pb. Constant shaking was applied in all elution stepsusing a horizontal shaker. Between each step, TK100 discs were rinsedtwo times on each side with water using a spray bottle to avoid carry-over.The elemental masses in each eluate fraction were determined afterICP-MS analysis and compared to those taken up by the TK100 discsto calculate the recovery in each fraction individually.The selectivity of TK100 membraneswas evaluated in an additionalmass balance experiment using the same immersion conditions as statedabove but further adding analytes, which interfere in subsequent MCICP-MS analysis: Ca (MDLs) and method quantificationlimits (MQLs) were calculated as three and ten timesthe standard deviation (SD) of the DGT blanks.Blanks were assessed usingTK100 DGT devices, which were treatedas the experimental devices but not exposed to the deployment medium.Method detection limits  provided by DGT Research Ltd. was used.27 Diffusion coefficients were corrected for temperatureusing the Stokes–Einstein equation.23A diaphragm diffusioncell was used to determine the diffusion coefficient, c = 500 μg L–1) and Pb (c = 50 μg L–1) at varying pH values or ionic strengths . Recovery of Sr and Pb was considered adequate at ratios of cDGT values to bulk solution concentration (csoln) between 0.85 and 1.15.29 Details of the solution preparation and analysis are provided inthe Supporting Information.The effect of pH andionic strength was evaluated by deploying triplicate TK100 DGT devicesfor 24 h in 2 L of well-stirred solutions spiked with both Sr (3 solution (c = 0.01 mol L–1) spikedwith Sr  at pH 6.6 ± 0.1. Afterremoval and DGT disassembly, TK100 membranes were subjected to single-stepelution in HCl (c = 6 mol L–1).Details of the solution preparation and analysis are provided in the Supporting Information.The analyte accumulation and possible isotopic fractionation overtime were investigated by deploying triplicate TK100 DGT devices forvarying times (2–24 h) in 3.5 L of well-stirred NaNOTable S2. To test competitive effects of Ca, three differentlevels of Ca competition were included, thus giving three differentimmersion solutions “A”, “B”, and “C”.The ionic strength was balanced at 0.013 mol L–1 using Na+ as the counterion. To further test whetherTK100 DGT sampling enables accurate isotope ratio measurement andsource tracing under competitive conditions, solutions “A”,“B”, and “C” were spiked with three differentSr isotope standard solutions. These solutions were prepared by mixingthe Sr(NO3)2 stock solution [δSRM 987(87Sr/86Sr) = −3.71± 0.25‰] with SRM 987 [δSRM 987(87Sr/86Sr) = 0.00 ± 0.37‰] togive three significantly different δSRM 987(87Sr/86Sr) values in solution . All solutions were preparedover two days under constant stirring to ensure equilibration beforeDGT deployment.TK100 DGT deviceswere deployed in triplicates for 24 h in 3.5 L of synthetic soil solutionsto evaluate the performance of the technique under competitive conditions.The elemental composition of the synthetic soil solutions was basedon typical literature values and is presented in 30 The two contaminated soils included an acidiccambisol with a loamy texture from Arnoldstein, AT (“AC”),and a calcic cambisol with a loamy texture from Mežica, SI(“MC”). These soils originate from former Pb miningareas and have been previously characterized by Lestan et al.31 The soils were air-dried, sieved to ≤2mm, and stored at room temperature in plastic zip-lock bags untiluse. Details on DGT deployment, soil solution sampling, and NH4NO3 (c = 1 mol L–1) extraction and sample preparation are provided in the Supporting Information.Three contrastingmineral soils(0–30 cm depth), one from an agricultural site and two frommetal-contaminated sites, were used. The agricultural soil was anacidic cambisol with a sandy loam texture from Siebenlinden, AT (“SL”),and has been previously characterized by Oburger et al.fu, over all mass balanceexperiments were 1.00 ± <0.01 for Sr and 0.98 ± 0.01for Pb. This demonstrated quantitative sampling of Sr and Pb by TK100membranes. The average elution recoveries, fe, were 0.95 ± 0.02 for Sr and 0.82 ± 0.04 for Pbafter single-step elution of TK100 membranes for 24 h in HCl (c = 6 mol L–1) . These values are consistent with previouslyreported Sr and Pb recoveries for TK100 column-type separations25 and indicate adequate elution recovery of Sr and Pb for isotoperatio measurements by MC ICP-MS.21 Thelow combined uncertainties (uc) on thesevalues demonstrate the high intermediate precision and reproducibilityof the analyte uptake and recovery .The average uptakeefficiencies, +, Mg2+, K+, and Ca2+.20 However, to date,no information is available on the adsorption of Rb and Hg, whichrepresent critical isobaric interferences in Sr and Pb isotope ratiomeasurements by MC ICP-MS.21 To determineif TK100 membranes also adsorb these elements, they were deployedin solutions containing Ca, Rb, and Hg along with Sr and Pb to ensurecompetitive conditions. Despite quantitative uptake of Sr (fu = 0.99 ± 0.01) and Pb (fu = 0.99 ± 0.01) (mean ± uc), significant co-uptake of Ca (fu = 0.91 ± <0.01), Rb (fu = 0.45± 0.01), and Hg (fu = 0.98 ±<0.01) was observed . Therefore, the ability of the TK100 membranes forchromatographic separation of Ca, Rb, and Hg from Sr and Pb was furtherinvestigated by applying different eluents. Application of HNO3 (c = 8 mol L–1) as a matrixwash in the first elution step as recommended for column-type TK100separations by Surman et al.20 resultedin high losses of Sr and Pb  and consequently inadequate recoveries for isotoperatio measurements.21 Using HCl (c = 0.01 mol L–1) in the first elutionstep, cobound Ca and Rb were effectively removed, while the lossesof both Sr and Pb were significantly reduced as compared to the originalelution scheme . However, Hg remained strongly retained (fe = 0.02 ± <0.01), indicating its high affinity forthe TK100 membrane as for the conventional Chelex gel.32 Hence, direct matrix-free sampling of Pb byTK100 DGT for isotope ratio analysis involving 204Pb islimited and eventually requires correction for residual 204Hg. In the second elution step, Sr (fe = 0.75 ± 0.03) and Pb (fe = 0.31± 0.01) were eluted in HCl (c = 2 mol L–1). Finally, residual Sr (fe = 0.01 ± <0.01) and the main part of Pb (fe = 0.56 ± 0.02) were eluted in the third elutionstep using HCl (c = 6 mol L–1).This elution scheme (3) was applied in all further experiments whereSr and Pb were sampled by DGT under competitive conditions. As Pbrecoveries in the individual eluate fractions were low, samples ofthe HCl eluates from elution steps two and three were combined (φ= 1) for MC ICP-MS analysis, giving an overall Pb recovery of 0.87± 0.02.TK100 has beenshown to be selective for Sr in the presence of majormatrix cations including Na–1 for Sr (n = 13) and 15.9± 2.9 ng disc–1 for Pb (n =10). This equates toaverage MDLs and MQLs of 0.21 and0.69 μg L–1 for Sr, respectively, and 0.38and 1.28 μg L–1 for Pb, respectively, fora DGT deployment of 24 h at 25 °C. The MDL forSr was thus three times lower than previously reported for ChelexDGT18 and also substantially lower thanthe minimum concentrations of Sr in soil solutions of highly weatheredtropical soils (1 μg L–1).33 However, the MDL for Pb was 19 times higherthan for Chelex DGT,18 indicating Pb contaminationof the TK100 membrane. While this is not relevant for the most importantapplication of the technique, i.e., Pb-contaminatedsoils where Pb concentrations in soil solutions of >100 μgL–1 are found,11 it maylimitPb measurements in natural noncontaminated soils where Pb concentrationsin soil solutions are often <1 μg L–1.34 Therefore, precleaning of TK100 membranes inHCl (c = 6 mol L–1) for 24 h wasperformed, which lowered the MDL for Pb by 1.5 timesdown to 0.25 μg L–1. Yet, the MDL was still 12.5 times higher than for Chelex DGT. This indicatedthat the main blank issue was related to the TK100 itself, as previouslyreported for the conventional crown-ether-based Sr spec resin.35 Further precleaning of the TK100 prior to embedmentin the PTFE membrane may be required to enable Pb measurements atultratrace levels.The average blank values were 1.32 ± 1.23 ngdiscD25) calculated from the regression line slope(0.74 ng s–1) was 6.17 × 10–6 ± 0.58 × 10–6 cm2 s–1. This value is 23% lower compared to the D25 of Sr in water (7.94 × 10–6 cm2 s–1),36 confirmingprevious works showing restricted diffusion of cationic solutes inAPA gels.38 However, Chang et al.17 reported a D25 for Sr in anAPA gel of 7.72 × 10–6 cm2 s–1, i.e., indicating almost free diffusionof Sr in the gel. The apparent difference is likely caused by a differentcomposition of the APA gel used by Chang et al.17 as compared to those that are now available commerciallyand routinely used. Changes in the manufacturing process of the agarosederivative cross-linker have resulted in gels with slightly smallerpore sizes,37 causing a more tortuous diffusionpathway through the polyacrylamide matrix and thus slower diffusivity.Diffusion coefficients of Sr and Pb in APA gels from 1 to 35 °Care provided in Table S4. These valueswere used for all calculations in this study.The massof Sr that diffusedwith time from the source into the receptor solution through the APAgel is shown in cDGT) were in excellent agreementwith those in solutions (csoln) in thepH range of 3.9–8.2 with an average cDGT/csoln ratio of 1.00 ±0.03 propane-1-sulfonicacid (MOPS) buffer exclusively used in the solution with pH 7.5, leadingto a substantial and continuing decrease of kinetically labile Pbin solution. It is possible that, at pH 7.5, deprotonation of theheterocyclic nitrogen atom in the morpholine ring of MOPS caused Pbcomplexation, as recently shown by isothermal titration colorimetry.39 Yet, it is unlikely that the dissociation rateconstant of these complexes is sufficiently low to significantly limitthe DGT uptake of Pb. More likely, Pb complexation by MOPS enhancedPb adsorption to the plastic container walls at pH 7.5, which hasbeen shown to also occur even at slightly acidic pH of 6.0.40 Although further research may be required tofully explain this observation, it is important to note that a directpH effect is implausible, as cDGT/csoln of Sr was close to 1 also at pH 7.5, whileboth Sr and Pb were sampled quantitatively under all other pH conditions.Concentrationsof Sr measured by TK100DGT  and viscosity effects (at I > 0.1 mol L–1) in diffusive gelsandsolutions, respectively.The  mol L–1 1, which c = 51.7 ± 0.4 mg L–1). Sr was chosen as atest element for the experiment given its generally higher abundancein natural soil solutions (Table S2), lowerbinding affinity of TK100,20 higher relativeisotopic mass difference, and a smaller isotopic natural-abundancevariation as compared to Pb, which implies that the determined accumulationand isotopic fractionation are conservative estimates that can beexpected to be applicable to Pb as well.Time-dependent analyte accumulationand possible isotopic fractionationduring incremental uptake were investigated by deploying TK100 DGTsfor up to 24 h in solutions with high levels of Sr (R2 > 0.99). TheDGT-measuredmasses, corrected for fe, were in excellentagreement with those predicted from mmeasured/mpredicted ratio of 0.99 ± 0.06(mean ± 1SD). The maximum Sr mass loading was904 μg (288 μg cm–2 at Ap = 3.14 cm2), which was still very close tothe predicted value of 862 μg and, notably, 1.6 times higherthan the total Sr capacity reported for a nonselective binding layercontaining the general cation-exchange resin AG50W-X8.17 The isotopic fractionation of 87Sr/86Sr, expressed as ΔSRM 987(87Sr/86Sr)DGT/soln DGT/soln values were not significantly different from zero with respect tothe uncertainty. The average expanded uncertainty  of δSRM 987(87Sr/86Sr)DGT values was 0.26‰,with MC ICP-MS analysis being the main contributor (85%). These resultsconfirmed that (i) there is sufficiently rapid uptake and high totalbinding capacity by the TK100 membrane to ensure zero-sink samplingof Sr, and (ii) that the TK100 DGT is an appropriate passive samplerfor the accurate and precise assessment of 87Sr/86Sr isotope ratios over the investigated time scale and conditionsused in this experiment.DGT/soln 4, with ic = 275 μg L–1) and Pb(c = 15 μg L–1) as well asdifferent levels of Ca to encompass environmentally relevant competitionconditions (Table S2). As for the time-seriesexperiment, Sr was chosen for evaluating the isotopic fractionation,given the higher relative isotopic mass difference and smaller natural-abundancevariation as compared to Pb and hence the increased susceptibilityto mass-dependent fractionation effects. The Sr concentrations and 87Sr/86Sr isotope ratios assessed by TK100 DGT andin the immersion solutions “A”, “B”, and“C” are shown in Table S5).The performanceof the TK100 DGT was tested in synthetic soil solutions spiked withSr (nditions S2. As focDGT/csoln ratios of Sr and Pb between 0.85and 1.15 (S5). Only at a Ca level of 159 mg L–1, which confines the upper range typically encountered in arablesoil solutions,34 the uptake of Sr wasslightly impaired, as evidenced by cDGT/csoln ratios approaching 0.85. It isnoteworthy that the performance for sampling Sr at the two upper levelsof Ca was substantially improved by precleaning the TK100 membranein HCl (c = 6 mol L–1) for 24 hfollowed by thorough rinsing and storage in water. Thereby, increased cDGT/csoln ratiosof Sr from 0.81 ± 0.14 to 0.94 ± 0.15 at 112 mg L–1 Ca and 0.72 ± 0.16 to 0.86 ± 0.14 at 159 mg L–1 Ca were obtained. The HDEHP in TK100 is a cation exchanger havingsome affinity for Ca at near-neutral pH, causing its uptake into theorganic phase. Precleaning of the TK100 membrane in HCl increasesprotonation of the organic phase, thus effectively lowering the HDEHPaffinity for Ca while maintaining high affinity for Sr and Pb evenat pH 2 and 3.20 Therefore, the proposedprecleaning procedure may be an effective strategy to improve theTK100 DGT binding efficiency for Sr when applied in soils with elevatedCa levels. Other elements with high affinity for TK100 20 are unlikelyto compete with Sr uptake, given their trace to ultratrace contentstypically present in natural soils.3The resultsshowed that competition effects are likely to be negligiblein typical soil solutions, with all and 1.15 2 and S5.Table S3; elution scheme 3) of TK100 membranes was applied afterDGT sampling to achieve separation of Sr and Pb from cobound matrixcations . The obtained average cation separation efficiency, expressedas the relative difference between the elemental mass concentrationratio in the synthetic soil solutions and in the DGT eluates, was≥96% for Sr and 100% for Pb (Table S6). Therefore, TK100 DGT sampling in combination with the selectivethree-step elution procedure in HCl enables direct in situ matrix separation of Sr and Pb and is thus beneficial for Sr andPb isotope ratio measurements by MC ICP-MS. Comparison of the isotopicdata for Sr sampled by TK100 DGT and in the standard solutions confirmedthat neither diffusive-based sampling nor selective elution causedfractionation of Sr isotopes, as ΔSRM 987(87Sr/86Sr)DGT/soln values remainedclose to zero with low expanded uncertainty for all deployments , as shown by cDGT/csoln ratios between 0.19 and 0.32. This indicatesthat the concentration of Sr in soil solution of the investigatedsoils is limited by diffusional transport and thus only partiallymaintained by resupply via desorption from soil solid phases.46 For Pb, the results of the concentration measurements by DGT weredistinctly different. As expected, cDGT values of Pb in the agricultural SL soil were significantly lowerthan in the metal-contaminated MC and AC soils 47 due to a more complex matrixof real soil DGT eluates (screening showed elevated levels of bariumand rare-earth elements). Pb eluates were analyzed without additionalseparation following dilution to 10 μg L–1 of Pb. To cope with this, we recommend that DGT eluates should alwaysbe screened to check if matrix loads are low enough to allow for directisotope ratio analysis by MC ICP-MS.The solution S7, as shδSRM 987(87Sr/86Sr)DGT, were significantlydifferent between all soils,ranging from 12.6‰ in SL to −1.43‰ in MC (δSRM 987(87Sr/86Sr)DGT in SL results fromweathering of the ancient granitic bedrock,48 releasing radiogenic 87Sr during soil formation. ForPb, only the metal-contaminated AC and MC soils were analyzed dueto a predominant contribution (>60%) of the procedural blank tothetotal Pb sampled by DGT in the arable SL soil. The obtained δSRM 981(207Pb/206Pb)DGT values of AC and MC were not significantly different, suggestingthat both methods access similar pools of Sr and Pb in soils. Thisis of high relevance for the application of the TK100 DGT techniqueas a tool for tracing the (geographic) origin of agricultural productsor sources of Sr and Pb contamination in the environment, where soilextraction using NH4NO3 has been indispensablein the past.10The resulting isotope ratiosof Sr measured in DGT eluates, 3‰ in MC 3, which ifferent3, indicaractions 3 and S8,This work has evaluated a new method for the accurate assessmentof the elemental and isotopic compositions of labile, bioavailableSr and Pb in soils with low uncertainties. Using the selectivity ofTK100 membranes together with the mechanistic sampling principle ofDGT, concomitant information on elemental resupply dynamics and naturalisotopic variations of Sr and Pb in soil was obtained for the firsttime. This method is a significant advancement in environmental analysisof Sr and Pb by allowing for simultaneous and time-integrated measurementsof labile Sr and Pb fractions with adequate elution recovery and quantitativematrix reduction already in the sampling step, thereby possibly facilitatingsample preparation for MC ICP-MS. The total capacity of the TK100DGT for binding Sr is very high compared to all existing Sr DGT techniques,and it performs effectively in the range of pH, ionic strength, andcation competition of environmental interest. Our results also showedthat neither diffusive-based sampling nor selective elution or massloading effects caused isotopic fractionation. Although detectionlimits for Pb were high compared to previous DGT techniques, thereis scope for substantial improvement by further precleaning of theTK100 itself, offering the potential for simultaneous analysis ofSr and Pb isotopes at ultratrace levels in the future. Applicationof the TK100 DGT to natural soils demonstrated its unique capabilityas a passive sampling tool for Sr and Pb analyses and source tracingfor further understanding of biogeochemical cycles of Sr and Pb insoils.


Serratia marcescens ATCC 27117 strain has been achieved to levels of 14 mg/L of culture within 24 h. Furthermore, environmentally friendly bromination of produced PG was used to afford both novel mono- and dibrominated derivatives of PG. PG and its Br derivatives showed anticancer potential with IC50 values range 0.62–17.00 µg/mL for all tested cancer cell lines and induction of apoptosis but low selectivity against healthy cell lines. All compounds did not affect Caenorhabditiselegans at concentrations up to 50 µg/mL. However, an improved toxicity profile of Br derivatives in comparison to parent PG was observed in vivo using zebrafish (Danio rerio) model system, when 10 µg/mL applied at 6 h post fertilization caused death rate of 100%, 30% and 0% by PG, PG-Br, and PG-Br2, respectively, which is a significant finding for further structural optimizations of bacterial prodigiosins. The drug-likeness of PG and its Br derivatives was examined, and the novel Br derivatives obey the Lipinski’s “rule of five”, with an exemption of being more lipophilic than PG, which still makes them good targets for further structural optimization.Prodigiosins (prodiginines) are a class of bacterial secondary metabolites with remarkable biological activities and color. In this study, optimized production, purification, and characterization of prodigiosin (PG) from easily accessible  Streptomyces spp., and Gram-negative bacteria, e.g., Serratia spp.  assay . BrieflyTo assess the effect of the treatment duration, exposure of MRC-5 and HCT116 cells to PG and its Br derivatives was observed after 24 h, 48 h, and 72 h and measured using the MTT assay, as described above. MRC-5 and HCT-116 cells treated for 24 h were additionally analyzed using DM IL LED Inverted Microscope  at 20× magnification.TM Annexin V Apoptosis Detection Kit APC  was used for the assessment of cellular integrity and the externalization of phosphatidylserine. MRC-5 cells were plated in a 6-well flat-bottom plate at a concentration of 2.5 × 105 cells per well, grown for 24 h in a humidified atmosphere of 95% air and 5% CO2 at 37 °C in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin . Each tested compound was added to the cells at IC50 concentration, and the incubation continued for another 24 h under the same conditions. Upon incubation, cells were collected, washed once with Dulbecco’s Phosphate-Buffered Saline , and stained with Annexin V FITC and propidium iodide (PI) under the manufacturer’s instructions. Briefly, MRC-5 cells were resuspended in 1 × Binding Buffer at 1–5 × 106 cells per mL and incubated with 5.00 µL Annexin V FITC for 15 min at room temperature in a final volume of 100 μL. After incubation, cells were washed and resuspend in 200 μL of 1 × Binding Buffer, 5.00 μL of Propidium Iodide Staining Solution was added, and incubation at room temperature continued for another 15 min. Immediately after staining, cells were analyzed using a CyFlow Space Partec flow cytometer, with Partec FloMax software .The eBioscienceCaenorhabditis elegans AU37) were obtained from the Caenorhabditis Genetics Center (CGC), University of Minnesota, Minneapolis, Minnesota, US. C. elegans AU37  was propagated under standard conditions, synchronized by hypochlorite bleaching, and cultured on nematode growth medium using E. coli OP50 as a food source, as described previously [C. elegans survival assay was carried out following the standard procedure with some modifications [2PO4, 6.0 g of Na2HPO4, 5.0 g of NaCl, and 1 mL of 1 M MgSO4∙7H2O in 1 L of water), 5% LB broth, and 10 μg/mL of cholesterol . The experiment was carried out in 96-well flat-bottomed microtiter plates  in the final volume of 100 μL per well. 25 μL of this suspension of nematodes (25–35 nematodes) were transferred to the wells of a 96-well microtiter plate, where 50 μL of the medium was previously added. Next, 25 μL of the solvent control (1% DMSO) or 25 μL of the concentrated tested compound solution was added to the test wells. Final concentrations of the compounds were 50.00, 25.00, 12.50, 6.25, 3.13, and 1.57 µg/mL. Subsequently, the plates were incubated at 25 °C for 2 days. The fraction of dead worms was determined after 48 h by counting the number of dead worms and the total number of worms in each well, using a Carl Zeiss™ Stemi 508 Stereomicroscope  at 40× magnification. The compounds were tested three times in each assay and each assay was repeated two times (n = 6). As a negative control experiment, nematodes were exposed to the medium containing 1% DMSO.Nematode roundworms (eviously . The C. ications . BrieflyDanio rerio) strain was obtained from the commercial supplier Pet Center, Belgrade, Serbia. Zebrafish embryotoxicity assay was performed according to general rules of the OECD Guidelines for the testing of chemicals [D. rerio) strain was kept under controlled environmental conditions  and fed regularly three times a day with commercially dry flake food supplemented with Artemia nauplii . Zebrafish embryos were produced by female and male adults mating in the ratio of 1:2. Obtained embryos were collected and washed from detritus and carefully handled. Only fertilized embryos were selected and distributed into 24-well plates  containing 10 embryos per well. Each well contained exactly 1 mL of water for embryos . For assessing lethal and developmental toxicity, embryos at the 6 and 20 h postfertilization (hpf) stages [w/v) tricaine solution , observed under a stereomicroscope  at 3.5× magnification, photographed, and killed by freezing at −20 °C for ≥24 h. All experiments involving zebrafish were performed in compliance with the European directive 2010/63/EU and the ethical guidelines of the Guide for Care and Use of Laboratory Animals of the Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia.Wild-type zebrafish  stages  were tre) stages ,84, and https://www.molinspiration.com, accessed on 26 May 2022, Slovensky Grob, Slovakia) was used for calculating physicochemical properties of the investigated compounds.Molinspiration tool  derivatives of PG. Although the biological activity of PG in the producing organism is not fully understood, it shows numerous biological functions in other organisms, including anticancer properties. Br derivatives of PG retained anticancer properties and showed favorable toxicity profiles in vivo, with satisfying pharmacokinetic profiles and drug-likeness properties.Low yield and high cost of extraction and purification still represent the bottleneck in microbial production of prodigiosin. In this study, we report straightforward fermentation of