IK(DR) amplitude in response to membrane depolarization in pituitary tumor (GH3) cells with an IC50 value of 5.71 and 1.32 μM, respectively. The current inactivation of IK(DR) elicited by 10-sec membrane depolarization became raised in the presence of roxadustatt. When cells were exposed to either CoCl2 or deferoxamine (DFO), the IK(DR) elicited by membrane depolarization was not modified; however, nonactin, a K+-selective ionophore, in continued presence of roxadustat, attenuated roxadustat-mediated inhibition of the amplitude. The steady-state inactivation of IK(DR) could be constructed in the presence of roxadustat. Recovery of IK(DR) block by roxadustat (3 and 10 μM) could be fitted by a single exponential with 382 and 523 msec, respectively. The roxadustat addition slightly suppressed erg-mediated K+ or hyperpolarization-activated cation currents. This drug also decreased the peak amplitude of voltage-gated Na+ current with a slowing in inactivation rate of the current. Likewise, in H9c2 heart-derived cells, the addition of roxadustat suppressed IK(DR) amplitude in combination with the shortening in inactivation time course of the current. In high glucose-treated H9c2 cells, roxadustat-mediated inhibition of IK(DR) remained unchanged. Collectively, despite its suppression of HIF prolyl hydroxylase, inhibitory actions of roxadustat on different types of ionic currents possibly in a non-genomic fashion might provide another yet unidentified mechanism through which cellular functions are seriously perturbed, if similar findings occur in vivo.Roxadustat (FG-4592), an analog of 2-oxoglutarate, is an orally-administered, heterocyclic small molecule known to be an inhibitor of hypoxia inducible factor (HIF) prolyl hydroxylase. However, none of the studies have thus far thoroughly investigated its possible perturbations on membrane ion currents in endocrine or heart cells. In our studies, the whole-cell current recordings of the patch-clamp technique showed that the presence of roxadustat effectively and differentially suppressed the peak and late components of  The hypoxia-inducible factor (HIF) pathway alters gene expression in response to low oxygen tension. Under normal conditions, HIF-specific prolyl hydroxylases (HIF-PHs) initiate the degradation of oxygen-sensitive HIF isoforms. ® in China), an analog of 2-oxoglutarate, is an orally administered, heterocyclic small molecule, known to inhibit HIF prolyl hydroxylases [Roxadustat  carbonyl]-glycine, Ai Rui Zhuooxylases ,9,10,11.V channels  has been noted to be associated with oxygen homeostats and induction of angiogenesis in certain types of tumors , erg-mediated K+ current [IK(erg)], and voltage-gated Na+ current [INa]) in pituitary tumor (GH3) cells. Moreover, earlier numerous studies have reported the effective regulations of HIF-1α expression present in heart-derived H9c2 myoblasts -glycine, C19H16N2O5, https://pubchem.ncbi.nlm.nih.gov/compound/Roxadustat) was acquired from Cayman , diazoxide, nonactin and PD-118057 were from Tocris , and deferoxamine (DFO), oxaliplatin and tetrodotoxin were from Sigma-Aldrich . Tissue culture media, fetal calf serum, horse serum, L-glutamine, penicillin-streptomycin, fungizone and trypsin/EDTA were obtained from Invitrogen Corp. , and other chemicals that include CdCl2, CoCl2, CsCl, CsOH, aspartic acid and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were of the best available quality, mostly at analytical grades.Roxadustat  or Ih, the patch electrode was filled with the following solution: 140 mM KCl, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer, pH 7.2. To measure INa, we replaced KCl inside the pipette solution with equimolar CsCl, and pH was adjusted to 7.2 with CsOH. To avoid possible contamination of Cl- currents, we substituted aspartate for Cl- ions inside the pipette solution. To measure IK(erg), bath solution was replaced with a high-K+, Ca2+-free solution containing 130 mM KCl, 10 mM NaCl, 3 mM MgCl2, and 5 mM HEPES-KOH buffer, pH 7.4, and the pipette solution contained 145 mM KCl, 2 mM MgCl2, and 5 mM HEPES-KOH buffer, pH 7.2. The composition of normal Tyrode’s solution was 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl3 pituitary tumor cells, obtained from the Bioresource Collection and Research Center , were grown in Ham’s F-12 medium supplemented with 15% horse serum (v/v), 2.5% fetal bovine serum (v/v), and 2 mM L-glutamine , IC50, and nH are the roxadustat concentration used, the concentration required for a 50% inhibition, and Hill coefficient, respectively; and, A or a represents the maximal fraction of total current (peak and late component) which is roxadustat sensitive or insensitive, respectively.To evaluate concentration-dependent inhibition of roxadustat on the amplitude of IK(DR) during cell exposure to different roxadustat concentrations, the relationships between the normalized amplitude of IK(DR) and the conditioning potentials obtained in the presence of 3 or 10 μM roxadustat were least-squares fitted with a modified Boltzmann function of the following form:Imax is the maximal peak amplitude of IK(DR) taken at the level of +50 mV, V1/2 the voltage at which half-maximal inhibition occurs, q the apparent gating charge of the inactivation curve, F Faraday’s constant, R the universal gas constant, T the absolute temperature, and a the residual fraction of the current.To determine the steady-state inactivation curve of n) indicating the cell numbers from which the results were collected. The paired or unpaired Student’s t-test and a one-way analysis of variance (ANOVA) followed by post hoc Fisher’s least-significance difference method, were implemented for statistical evaluation of differences among means. If normality underlying ANOVA was presumably violated, we attempted to use the non-parametric Kruskal–Wallis test. Statistical analyses were performed using the SPSS 20 statistical software package . Statistical significance was determined at a p-value of <0.05.Linear or non-linear curve-fitting  to data sets was performed with the goodness of fit by using different maneuvers such as Microsoft Solver function embedded in Excel 2016 (Microsoft) and 64-bit OriginPro 2016 program (OriginLab). The averaged results are presented as the mean±standard error of the mean (SEM) with sample sizes (IK(DR) in GH3 and H9c2 cells. The modification of aconitine on IK(DR) was reported to aberrantly prolong action potential in heart cells, in combination with the emergence of polymorphic ventricular tachycardia. Therefore, whether roxadustat caused any serious complications in heart function needs to be determined as an imperative, despite a previous study showing little or no occurrence of serious adverse cardiac events during roxadustat treatment. Nonetheless, our study reveals another unidentified but important aspect which needs to be given due consideration, despite the inhibitory actions of either roxadustat or other structurally similar compounds on the activity of HIF prolyl hydroxylase inside the cell. Such actions on these ionic currents are of particular clinical, pharmacological or toxicological relevance.In the present study, we found that roxadustat was effective at modifying the amplitude and gating of


In this paper, imitation basalt compounds using red mud, fly ash or coal gangue as raw materials were designed and prepared with the help of thermodynamic calculations. Thermodynamic calculations were used to obtain the suitable chemical composition. Then, the imitation compounds were prepared and their phase/compositions were analyzed. Finally, their high-temperature melting performance and crystallization ability were evaluated. The results show that the characteristic temperature and crystallization ability of the imitation basalt compounds were similar to those of basalt. Moreover, the viscosity of red mud imitation basalt compound approached the viscosity of basalt with the increase in temperature. This work suggests that red mud, fly ash and coal gangue can be mixed with quartz and other source materials to produce imitation basalt fiber. Therefore, thermodynamic calculation is an effective method to design and prepare high-performance imitation basalt compounds.  Continuous basalt fiber is formed by drawing the basalt melt (at a melting temperature of 1450–1500 °C) with a wire drawing machine. The production process of basalt fiber determines that the production process produces less waste and less environmental pollution. The product is directly degradable in the environment without any hazard and is therefore an environmentally friendly material. Due to its excellent mechanical properties, resistance to chemical erosion, resistance to high temperature and environmental resistance . However2, Al2O3, CaO, Fe2O3 and MgO; its detailed composition is shown in Basalt is a natural volcanic rock mainly consisting of SiO2O3, SiO2, CaO and Fe2O3. During the past century of alumina production, about 2.7 billion tons of red mud was produced [Red mud is a toxic waste that is discharged from the alumina production process. It cannot be disposed easily due to its alkali content which can contaminate the environment. Its main components are Alproduced . Howeverproduced , which l2O3 and SiO2. At present, the cumulative amounts of fly ash and coal gangue in China have reached 1.1 billion tons and 7 billion tons, respectively [Coal gangue is a solid waste generated during coal mining and coal washing, while fly ash is a solid waste generated during thermal power generation. The main components of both coal gangue and fly ash are Alectively ,11. Such2 and Fe2O3 in Bayer red mud enhanced the chemical resistance of the glass. Furthermore, Li, Luo et al. [For example, Luan, Li et al.  used flyo et al.  preparedo et al. ,16. TheyNumerous studies have shown that red mud, fly ash, and coal gangue can be used to produce aluminosilicate glass, which is significant because basalt fiber is a type of aluminosilicate glass ,19,20,212O3, and other raw materials to prepare the imitation basalt compounds. Finally, experiments were conducted to investigate the differences in the high-temperature melting performance and crystallization ability of basalt and imitation basalt compounds. This research extends the application of red mud to produce basalt fiber and also provides guidance for the production of imitation basalt fiber from fly ash and coal gangue.Considering the above background, this paper attempts to make full use of red mud, fly ash, and coal gangue to develop high-performance imitation basalt compounds. For this purpose, thermodynamic calculations were employed to obtain the chemical compositions of imitation basalt compounds with excellent high-temperature melting performance and low crystallization ability. Then, red mud, fly ash, and coal gangue were mixed with quartz, Al2, Al2O3, CaO, Fe2O3 and MgO, with small amounts of K2O, Na2O and TiO2. However, Fe2O3 and TiO2 play a key role in the crystallization process of basalt, which strongly affects the performance of continuous basalt fiber [2-Al2O3-CaO-Fe2O3-MgO-TiO2-O2, as shown in 2O3, and TiO2 was the same as that in the basalt sample. Since the points near the phase boundary are not easily crystallized during the cooling process, three points a (0.679 0.106 0.215), b (0.681 0.121 0.198), and c (0.698 0.085 0.217) near the phase boundary in the ternary phase diagram were selected according to the chemical composition of basalt. The sum of the mass ratios of SiO2, Al2O3, and CaO in the imitation basalt compounds is the same as that of the basalt sample. According to the coordinates of the three points a, b, and c in the ternary diagram of SiO2-Al2O3-CaO-Fe2O3-MgO-TiO2-O2, the chemical compositions of the imitation basalt compounds were calculated. 2O and Na2O as that in the basalt sample.The main chemical components of basalt are SiOlt fiber . TherefoThe high-temperature viscosities of basalt and three imitation basalt compounds were calculated by FactSage software. First, the Equilb module of FactSage software was adopted in order to obtain the uniform chemical compositions of the liquid phase in the system under thermodynamic equilibrium conditions. Then, the viscosity module of the FactSage software was applied to calculate the liquid phase chemical compositions obtained from the Equilb module to determine the liquid phase viscosity under the corresponding conditions. If a solid phase existed in the system, the Einstein–Rosce equation was adopted in order to calculate the liquid phase viscosity obtained from the viscosity module, and the solid phase content obtained from the Equilb module was used to obtain the viscosity of the solid–liquid mixture. The calculated viscosities were plotted as a function of temperature, as shown in The high-temperature viscosity of fly ash imitation basalt compound and coal gangue imitation basalt compound were calculated, and the obtained viscosities were plotted as a function of temperature, as shown in The basalt used in the present study was collected from Shandong Juyuan Basalt Fiber Co., Ltd., Zhangqiu, China; the red mud was obtained from Zunyi Alumina Plant, Zunyi, China; the fly ash and coal gangue were from Lingshou County Soil and Mineral Products Processing Factory, Shijiazhuang, China; quartz (AR), alumina (AR), ferric oxide (AR), magnesium oxide (AR), calcium carbonate (AR), sodium carbonate (AR), potassium hydroxide (UR), and titanium dioxide (AR) originated from Baihua Mall . In order to remove residual moisture, the raw materials were dried at a temperature of 393 K for 24 h. Following the drying processes, red mud, quartz, alumina, ferric oxide, magnesium oxide, calcium carbonate, potassium hydroxide, and sodium carbonate were, respectively, mixed in a weight ratio of 22.1:48.1:8.8:3.1:5.0:10.0:1.2:3.0 to prepare the red mud imitation basalt compound. Fly ash 1, quartz, alumina, ferric oxide, magnesium oxide, calcium carbonate, potassium hydroxide, sodium carbonate, and titanium dioxide were, respectively, mixed in a weight ratio of 33.0:41.6:0.1:6.1:5.1:11.1:1.4:5.6:0.3 to prepare the fly ash 1 imitation basalt compound. Fly ash 2, quartz, ferric oxide, magnesium oxide, calcium carbonate, potassium hydroxide, sodium carbonate, and titanium dioxide were, respectively, mixed in a weight ratio of 35.0:33.6:6.4:5.2:14.3:1.5:5.6:0.3 to prepare the fly ash 2 imitation basalt compound. Coal gangue, quartz, ferric oxide, magnesium oxide, calcium carbonate, potassium hydroxide, sodium carbonate, and titanium dioxide were, respectively, mixed in a weight ratio of 36.2:34.8:6.5:5.2:15.5:1.5:5.6:0.4 to prepare the coal gangue imitation basalt compound. The prepared imitation basalt compounds have the same chemical composition as the imitation basalt compound b in The chemical compositions of basalt, red mud, fly ash 1, fly ash 2, coal gangue, quartz, alumina, and the resulting imitation basalt compounds were analyzed by a Zetium X-ray fluorescence spectrometer .The phase components of basalt red mud, fly ash 1, fly ash 2, and coal gangue were analyzed by X’Pert PRO MPD X-ray diffractometer . The softening temperature, hemispherical temperature and flow temperature of the samples were tested by a MTLQ-RD-3 melting point meter , and the morphology changes of the samples were recorded.The high-temperature viscosity of basalt and red mud imitation basalt compound were measured by RSV-1600 high-temperature viscosity tester , and measured viscosity values were plotted as a function of temperature. To study their crystallization ability, the samples were first heated from room temperature to 1500 °C by a GSL-1700 high-temperature tube furnace ; then, the temperature was maintained at 1500 °C for 1 h and then cooled to 700 °C at a rate of 5 °C/min. Finally, the samples were cooled to room temperature in the furnace. The crystallization degree of samples was analyzed by X’Pert PRO MPD X-ray diffractometer. The microstructures of the samples after high-temperature treatment were characterized by EM-30 Pluse scanning electron microscope .X-ray diffraction patterns of the samples are shown in (1) The as-prepared imitation basalt compounds exhibited excellent high-temperature melting performance and low crystallization ability, indicating that red mud, fly ash, and coal gangue with different chemical compositions and phase components can be used to produce imitation basalt fiber.(2) Using thermodynamic calculations to obtain the chemical composition of imitation basalt compounds with desired properties is an effective method for the preparation of high-performance imitation basalt compounds.(3) The high-temperature melting performance and crystallization ability of the imitation basalt compounds are strongly dependent on their chemical compositions.


Background: Mucosal IL-13 Receptor alpha 2 (IL13RA2) mRNA expression is one of the best predictive markers for primary non-responsiveness to infliximab therapy in patients with inflammatory bowel disease (IBD). The objective of this study was to understand how IL-13Rα2, a negative regulator of IL-13 signaling, can contribute to IBD pathology.Methods:IL13RA2 knockout (KO) and wild type (WT) mice were exposed to dextran sodium sulfate (DSS) in drinking water to induce colitis. Furthermore, mucosal biopsies and resection specimen of healthy individuals and IBD patients before the start of anti-tumor necrosis factor (anti-TNF) therapy were obtained for immunohistochemistry and gene expression analysis.Results: After induction of DSS colitis, IL13RA2 KO mice had similar disease severity, but recovered more rapidly than WT animals. Goblet cell numbers and mucosal architecture were also more rapidly restored in IL13RA2 KO mice. In mucosal biopsies of active IBD patients, immunohistochemistry revealed that IL-13Rα2 protein was highly expressed in epithelial cells, while expression was restricted to goblet cells in healthy controls. Mucosal IL13RA2 mRNA negatively correlated with mRNA of several goblet cell-specific and barrier genes, and with goblet cell numbers.Conclusions: The data suggest that IL-13Rα2 on epithelial cells contributes to IBD pathology by negatively influencing goblet cell recovery, goblet cell function and epithelial restoration after injury. Therefore, blocking IL-13Rα2 could be a promising target for restoration of the epithelial barrier in IBD. As this holds true both in UC and in CD, two diseases mediated by different combinations of cytokines (IL13RA2 expression reflects an increased TNF burden in non-responders (Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract with a relapsing and remitting course. They are thought to result from an excessive immune response toward the intestinal and colonic microbiota in genetically susceptible hosts . Currentytokines , these rsponders . HoweverIL13Rα2 transcript and exclusively express this receptor as a transmembrane molecule  which, after ligand binding, activate the transcription factor STAT-6 . In rodemolecule . Howevermolecule –14.IL13RA2 expression can contribute to the pathology of IBD. First, we performed experimental studies with IL13RA2 KO and WT mice subjected to experimental colitis. Next, we determined the localization of the IL-13Rα2 protein in human colonic biopsies by immunohistochemistry. Based on the murine findings and because it is known that IL-13 influences goblet cells, we further correlated IL13RA2 expression with goblet cell specific genes, barrier genes and goblet cell numbers in IBD.The present study aimed to identify how IL13RA2 knock out (KO) mice backcrossed more than 10 times in the Balb/c background were obtained from Pfizer . Both IL13RA2 KO and wild type (WT) Balb/c mice were bred in the specific pathogen free area of the animal care facility of the KU Leuven, Belgium. Acute colitis was induced by administration of dextran sodium sulfate  in drinking water (3%) for 7 days, followed by a recovery period. Mice were sacrificed by injection of pentobarbital at day 9 or 12 for evaluation of markers of disease activity and inflammation. Colons were harvested, weighted and measured from the ileocecal junction to the anus. A macroscopic score of inflammation was given as previously described . Three sections per mouse were blindly and independently scored by a post-doctoral researcher (CP) and by an experienced IBD pathologist (GDH). The following five characteristics were scored cumulatively. Architectural disturbance, based on irregularity of the mucosal surface, crypt distortion and branching, loss of glands, and fraying of the muscularis mucosae ; goblet cell depletion ; epithelial defects ; neutrophil infiltration ; mononuclear cell infiltration .2O2 in methanol. Epitope retrieval was performed by heating the slides for 30 min in Tris/EDTA buffer (pH 9) at 98°C. Sections were then incubated with the anti-human IL-13Rα2 mouse monoclonal antibody clone ab55275  at a concentration of 1 μg/ml for 30 min. IL-13Rα1 protein was localized using anti-human IL-13Rα1 rabbit polyclonal antibody ab79277 (Abcam) at a concentration of 10 μg/ml. The Dako REALTM EnvisionTM Detection System kit  was used for visualization of bound primary antibody according to the manufacturer's instructions. Formalin-fixed, paraffin-embedded surgical biopsies of an ovarian serous adenocarcinoma served as positive controls for IL-13Rα2  in R version 3.5.0 , whereafter outliers were detected and removed based on cluster analysis  of inflamed mucosal biopsies of IBD patients prior to their first infliximab administration were analyzed. Patient characteristics at baseline were previously described , 18. Rawanalysis . MicroarP-values were obtained using two-tailed Mann-Whitney U testing. Differences were considered statistically significant at p < 0.05. Human data analysis was performed in R . Correlations between IL13RA2 expression and goblet cell related genes and numbers were analyzed using bivariate two-tailed correlation tests.Murine data analysis was performed with GraphPad Prism 5 . Data are represented as medians, and IL13RA2 KO and WT mice by DSS administration. At day 9, weight loss was most severe in both strains, reflecting the time point of strongest inflammation. At day 12 (recovery phase), most mice had recovered their initial weight despite persistent inflammatory features in the colon. Both strains developed colitis and equally lost weight (p = 0.07 at both day 9 and 12)  , the colon length (p < 0.001) and colon weight (p = 0.01), reflecting that inflammation resolved more rapidly in IL13RA2 KO mice.Acute colitis was induced in ) Figure . The inf Figures . In contIL13RA2 KO and WT mice at day 9 (p = 0.82), but it was significantly lower in IL13RA2 KO mice at day 12 (p = 0.02) . More specifically, the characteristics that were different between the two strains of mice on day 12 were the architectural changes (p = 0.009), the goblet cell depletion (p = 0.01) and the infiltration of neutrophils (p = 0.03)  Figure . At day ) Figure . Taken tIL13RA2 is highly expressed in the mucosa of IBD patients, and more so in anti-TNF non-responders    Figure . Thus, tCDN1, DSG3) or downregulation  of particular genes in active IBD compared to healthy individuals  and DSG3 expression , whereas IL13RA2 correlated negatively with CLDN8 , OCLDN , MEP1A , MAGI1 , and MARVELD2 .In general, these murine and human findings suggest a link between IL13RA2 and mucosal barrier function. Based on previous findings demonstrating an intestinal epithelial barrier dysfunction in IBD, with up-  in complex with TMEM219 (the chitosome) or Galectin-3 , can regulate a variety of cellular and tissue responses , 44, 45.IL13RA2 expression correlates negatively with numbers of goblet cells, as well as with the expression of goblet cell transcription factors and goblet cell function, suggesting that high IL13RA2 expression impairs goblet cell development. The role of IL-13Rα2 is further supported by murine data showing a more rapid recovery of IL13RA2 KO mice compared to WT mice after DSS induced colitis, with a more rapid restoration of goblet cells. In anti-TNF therapy non-responder patients in whom there is increased IL13RA2 expression , collecting serum, biopsies and clinical characteristics among other items. All murine studies were approved by the local ethical committee for animal experimentation of the KU Leuven(P013/2011).BV: contributed to the acquisition of data, analysis and interpretation of data, drafting of the manuscript, and statistical analysis. CP: study concept and design, acquisition data, analysis and interpretation of data, and drafting of the manuscript. GDH: acquisition data, analysis and interpretation of data, and critical revision of the manuscript for important intellectual content. JC: acquisition data, analysis, and interpretation of data. BC: interpretation of data. GVA: study concept and design, interpretation of data, and critical revision of the manuscript for important intellectual content. MF and SV: acquisition of the data, interpretation of data, and critical revision of the manuscript for important intellectual content. JLC: study concept and design, interpretation of data, and critical revision of the manuscript for important intellectual content. CB: study concept and design, acquisition of data, analysis and interpretation of data, material support, drafting of the manuscript, and study supervision. All authors agreed with the final version of the manuscript. CB: guarantor of the manuscript.BV received financial support for research from Pfizer; lectures fees from Abbvie, Ferring, Takeda Pharmaceuticals, Janssen and R Biopharm; consultancy fees from Janssen. GDH received consultancy fees from Centocor and Takeda. GVA received financial support for research from Abbott and Ferring Pharmaceuticals; lecture fees from Janssen, MSD and Abbott; consultancy fees from PDL BioPharma, UCB Pharma, Sanofi-Aventis, Abbott, Abbvie, Ferring, Novartis, Biogen Idec, Janssen Biologics, NovoNordisk, Zealand Pharma A/S, Millenium/Takeda, Shire, Novartis, and Bristol Mayer Squibb. MF received financial support for research from Takeda and Janssen; lecture fees from Ferring, Boehringer- Ingelheim, Chiesi, Merck Sharpe andamp; Dohme, Tillotts, Janssen Biologics, AbbvieTakeda, Mitsubishi Tanabe, Zeria; consultancy fees from Abbvie, Boehringer-Ingelheim, Ferring, Merck Sharpe andamp; Dohme, and Janssen Biologics. SV received financial support for research from MSD, Abbvie, Janssen and UCB Pharma; lecture fees from Abbott, Abbvie, Merck Sharpe andamp; Dohme, Ferring Pharmaceuticals and UCB Pharma; consultancy fees from Pfizer, Ferring Pharmaceuticals, Shire Pharmaceuticals Group, Merck Sharpe andamp; Dohme, and AstraZeneca Pharmaceuticals. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glioblastoma multiforme (GBM) is a type of brain tumor that is notorious for its aggressiveness and invasiveness, and the complete removal of GBM is still not possible, even with advanced diagnostic strategies and extensive therapeutic plans. Its dismal prognosis and short survival time after diagnosis make it a crucial public health issue. Understanding the molecular mechanisms underlying GBM may inspire novel and effective treatments against this type of cancer. At a molecular level, almost all tumor cells exhibit telomerase activity (TA), which is a major means by which they achieve immortalization. Further studies show that promoter mutations are associated with increased TA and stable telomere length. Moreover, some tumors and immortalized cells maintain their telomeres with a telomerase-independent mechanism termed the “alternative lengthening of telomeres” (ALT), which relates to the mutations of the α-thalassemia/mental retardation syndrome X-linked protein (ATRX), the death-domain associated protein (DAXX) and H3.3. By means of the mutations of the telomerase reverse transcriptase (TERT) promoter and ATRX/DAXX, cancers can immortalize and escape cell senescence and apoptosis. In this article, we review the evidence for triggering GBM cell death by targeting telomerase and the ALT pathway, with an extra focus on a plant-derived compound, butylidene phthalide (BP), which may be a promising novel anticancer compound with good potential for clinical applications. Despite their low incidence rate  . HoweverPatients with brain tumors may present with various signs and symptoms, such as severe, persistent, or recurrent headache; nausea and vomiting; papilledema; seizures; focal neurological and cognitive impairments, such as difficulty in speaking or thinking of words; disturbed vision, hearing, smell, or taste; weakness or paralysis in part of the body; loss of balance; general irritability; drowsiness or a change in personality; flashbacks; and loss of memory . In geneTERC), telomerase reverse transcriptase (TERT), epidermal growth factor receptor (EGFR), coiled-coil domain containing 26,cyclin-dependent kinase inhibitor 2B, pleckstrin homology such as domain family B member 1, tumor protein p53(TP53), and the regulator of telomere elongation helicase 1 (RTEL1) [TERC, TERT, and RTEL1) contain genes involved in telomere maintenance. Moreover, recent publications demonstrate that acquired somatic mutations in TERT and α-thalassemia/mental retardation syndrome X-linked protein (ATRX) can affect telomere maintenance in tumor cells and are important in glioma development and prognosis [In the past, only a few GBM risk factors were identified as being linked to environmental stimuli, including poor diet, smoking, cellular phones usage or exposure to an electromagnetic field, severe head injury, occupational risk factors and pesticide exposure ,21,22,23 (RTEL1) ,39,40,41rognosis ,43,44,45The etiology of GBM is unknown. GBM can be divided into primary and secondary GBMs, based on clinical or histological evidence. Primary GBMs develop rapidly de novo in older patients without clinical or histological evidence of less malignant precursor lesions. Secondary GBM develops more slowly, from low-grade diffuse astrocytoma or anaplastic astrocytoma, in young patients with a significantly better prognosis. Histologically, primary and secondary GBMs are similar, but they differ in their genetic profiles, including the mutations of isocitrate dehydrogenase (IDH), the co-deletion of chromosomes 1p and 19q, mutations of H3F3A, telomerase, TERT, and ATRX.IDH1) in 12% of patients with GBM [IDH1 mutations often have mutations of isocitrate dehydrogenase 2 (IDH2) [+)-dependent, catalyze oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), and reduce oxidized nicotinamide adenine dinucleotide (NAD+) or NADP+ to NADH or NADPH. IDH1, localizing in the cytoplasm and peroxisomes, involves cellular metabolism and protection from reactive oxygen species and radiation [DH1 and IDH2 mutations are mono-allelic, somatic, and missense changes. Mutations in IDH1 commonly affect R132, which is the binding site for isocitrate [IDH2 only affect R172 and R140 [IDH1 and IDH2 are very rare in childhood GBM [To elucidate genetic alterations in GBM patients with varying prognoses or responses to specific targeted therapies and to identify subgroups of GBM patients for a better histopathological classification, an integrated genomic analysis was used to identify mutations inisocitrate dehydrogenase 1(with GBM . It was 2 (IDH2) . The str2 (IDH2) . IDH1 anadiation ,50. IDH2ocitrate . Mutatioand R140 ,51. Mutaand R140 . Excessiand R140 . Both muhood GBM , suggestFUBP1) on chromosome 1p and the capicua transcriptional repressor (CIC) on chromosome 19q [FUBP1 expresses a single-stranded DNA-binding protein that can bind to several DNA regions, which harbor the far upstream element (FUSE) that is localized in the upstream of c-Myc. One function of FUSE is to regulate c-Myc in undifferentiated cells [The complete deletion of chromosomes 1p and 19q is common in oligodendrogliomas and occur in 50–70% of both low-grade and anaplastic tumors ,56,57,58some 19q ,60,61. Ted cells . CIC, a ed cells . Two clied cells ,65. Howeed cells ,67,68.H3F3A at lysine 27 is at or near critical regulatory histone residues, therefore, the alternations of mutant K27M on genes, which should be silent, produce widespread aberrant DNA methylation and deregulation of gene expression, impede physiological differentiation and to drive cell transformation [Inside the nucleus, DNA, RNA and proteins form chromatin, which packs the DNA to a smaller volume and prevents the long DNA strands from being tangled. The structure of the chromatin is like “beads on a string”. The nucleosome is the “bead”, which is a basic unit of chromatin. Histones, nuclear proteins, can store DNA, modulate chromatin structure, impact gene expression, and regulate almost all DNA metabolic processes through post-translational modification, which includes methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation . Each nuormation . Furtherormation . The mutormation . Anotherormation . H3F3G34ormation  and upreormation .A telomere is a capping structure located at the end of a linear chromosome. As most prokaryotes have circular chromosomes, they do not have telomeres; on the other hand, telomeres in vertebrates consist of a region of repeats of the six-nucleotide sequence TTAGGG at the ends of chromosomes, which themselves carry the complementary DNA strand sequence, AATCCC. In humans, the TTAGGG sequence repeats in tandem approximately 3000–20,000 times ,79. The TERT promoter mutations (C228T and C250T) [TERT, which cause TERT activation to increase TA to elongate telomere length [Cell division leads to progressive telomere shortening, resulting in cell senescence; however, the shortening of telomeres can be counteracted by telomerase . Telomerd C250T)  in the ad C250T) ,121, incd C250T) . The twoe length ,123, leae length ,125,126.e length ,123 and e length ,128.TA was detected in 76% of cervical cancer cases , 54% of TERT promoter frequently harbor mutations of ATRX to activate ALT [α-thalassemia X-linked intellectual disability (ATRX) syndrome is characterized by distinctive craniofacial features, genital anomalies, severe developmental delays, hypotonia, intellectual disability, and mild-to-moderate anemia secondary to α-thalassemia .The ATRXvate ALT . Yang etvate ALT , suggestThere are several promising novel anticancer compounds with good potential for clinical applications. For example, the inhibition of mutant IDH may potentially have anticancer effects. Accordingly, the Food and Drug Administration (FDA) has approved the mutant IDH1 inhibitor, ivosidenib , and IDHH3F3A mutation with loss of ATRX and ALTis reported [Currently, the investigated agents targeting telomerase for cancer treatments are considered ineffective in ALT-dependent cancers or cancers adapting ALT mechanisms. Hence, to achieve effective tumor cell senescence and apoptosis by blocking telomere lengthening, not only should the telomerase-associated pathway be targeted, but the proper functioning of the ATRX/DAXX/H3.3 complex should be retained. Given the fast-paced development of genome editing technology, gene therapies targeting ATRX are considered to be among the potential treatments for ALT-positive cancers . Additioreported , and mutreported , and DNAreported , these freported , panobinreported , cyclic reported , andH3-Kreported  may be eAngelica sinensis (Oliv.) Diels (AS), commonly known as dong quai, is used for the treatment of gastric mucosal damage, hepatic injury, chronic glomerulonephritis, impaired myocardial blood flow, and menopause in traditional Chinese medicine [AS, butylidenephthalide (BP), inhibits the proliferation of human GBM through the down-regulation of TERT and the consequent reduction in TA, leading to tumor senescence [β-galactosidase [Natural products have a long history of wide use as cancer treatments in China. Traditional Chinese medicines typically make use of plants or their derivatives and are usually considered alternative therapeutic options or palliative treatments, especially in the context of cancer treatment. However, there is an increasing amount of evidence supporting the use of plant extracts in direct and primary cancer treatments. For example, medicine , and hasmedicine ,182,183.nescence . Meanwhinescence . In resetosidase . In DBTRtosidase . In sum,GBM can grow in all cortical areas, the brainstem, and the spinal cord. Patients with GBM may rapidly present with various signs and symptoms. It is so aggressive and invasive that even with maximal surgical resection plus radiotherapy with concomitant or subsequent chemotherapy, GBM remains an incurable tumor and a median survival of less than 2 years from diagnosis ,15. This


As a result of different abiotic stresses, especially those resulting from global warming, K+ fluxes and plant distribution of this ion are disturbed. The hormone abscisic acid (ABA) is a key player in responses to these climate stresses. It triggers signaling cascades that ultimately lead to modulation of the activities of K+ channels and transporters. After a brief overview of transcriptional changes induced by abiotic stresses, this review deals with the post-translational molecular mechanisms in different plant organs, in Arabidopsis and species of agronomical interest, triggering changes in K+ uptake from the soil, K+ transport and accumulation throughout the plant, and stomatal regulation. These modifications involve phosphorylation/dephosphorylation mechanisms, modifications of targeting, and interactions with regulatory partner proteins. Interestingly, many signaling pathways are common to K+ and Cl−/NO3− counter-ion transport systems. These cross-talks are also addressed.As the main cation in plant cells, potassium plays an essential role in adaptive responses, especially through its involvement in osmotic pressure and membrane potential adjustments. K Also, an increase in irrigation use leads to soil salinization. Drought and heat stresses, especially when combined, are responsible for significant losses in agriculture [Arabidopsis thaliana also indicate that ABA plays a role in the adaptation to high temperatures [Global warming is impacting crop yield and quality. Increases in atmospheric COiculture ,2,3. Dificulture . Abscisiiculture ,5,6,7 aneratures ,9.+ and anions across cellular membranes typically occur at the end of the biological responses to environmental stimuli.In plants, the synthesis of the phytohormone ABA, which probably occurs mainly in vascular tissues , is indu+ fluxes is a well-known response to ABA drought and salt stress. K+ is the main cation in plant cells, essential for plant growth and adaptation to the environment [+ because the latter affects the water H-bonding at the protein surface more than K+ [+, which is involved in cell turgor, can to some extent be replaced by other osmotica [+ is involved in nasties, including stomatal movements [+ also displays biochemical properties. It is involved in the direct or indirect activation of enzymes involved in metabolic processes, such as carbon metabolism and protein synthesis [+ in mesophyll CO2 conductance, chloroplast organization, improvement of Rubisco activity, and translocation of photoassimilates in the phloem [+ limits reactive oxygen species (ROS) production that leads to photoinhibition [+ homeostasis even under stressful conditions is, therefore, a necessity for plant growth and adaptation [+ uptake and, thus, exacerbates the effects of K+ deficiency [+ translocation to the xylem [+-mediated stomatal closure for preventing dehydration. Salinity also results in root and shoot decline in K+, salt tolerance being correlated to the ability to save K+, and to the cytosolic K+/Na+ ratio [+ homeostasis is particularly critical for plant tolerance to drought and salt stresses [Modulation of Kironment ,14. Due ironment . Its con than K+ . In contosmotica ,18. As aovements . K+ alsoynthesis . In K-dee phloem . Under hhibition . Maintaiaptation . Droughtficiency . It alsohe xylem , thus mahe xylem , and tria+ ratio ,24. Indestresses ,23 and hstresses .+ transport systems are targets of signaling pathways, leading to environmental responses such as K+ uptake, translocation, and stomatal movements. Multiple channels and transporters were identified in plants [+ transport systems which drive K+ fluxes as essential end responses to different abiotic stresses and ABA signaling cascades, and on their relationships with those regarding mineral anion  transports.Kn plants . In thisThe Shaker channels: K+ channels of the Shaker family mediate the major K+ fluxes at the plasma membrane [+ ions. Each subunit is made up of a short cytosolic N-terminal sequence, a membrane part containing the pore-forming domain with the K+ selectivity filter, and a large C-terminal intracytoplasmic part including a cyclic-nucleotide-binding domain, an ankyrin repeat protein–protein interaction domain, and a conserved hydrophobic and acidic (KHA) C-terminal end. In Arabidopsis thaliana, Shaker channels are encoded by a family of nine genes, which are grouped into five subfamilies , KAT1, KAT2 (K+Arabidopsis transporters 1 and 2), and AtKC1  subunits assemble as Kin channels, whereas SKOR (stelar K+ outward rectifyer) and GORK (guard cell outward rectifying channel) subunits form Kout channels [+ currents are likely to correspond to AKT1/AtKC1 heteromers rather than AKT1 homotetramers [+ from the soil (even from K+ solutions as dilute as 10 µM) and its secretion into the xylem, transport of solutes in the phloem, stomatal movements, and cell elongation . Theferences ,29,30. Schannels . A notabchannels . At the channels ,33,34. Petramers . Shaker etramers . They arongation . Members+ channels)TPKs (Two-pore K constitute the other group of K+-selective channels. This family comprises six members in Arabidopsis. Except for KCO3 (Ca2+ activated outward rectifying K+ channel 3), which could be an inactive channel, they are dimers of two-pore domain-subunits, composed of four transmembrane segments with two pore domains including the K+-selectivity motif GYGD. Of these, only TPK4 (two-pore K+ transporter 1) is targeted to the plasma membrane [2+ and cytoplasmic acidification, is an open rectifier channel. This channel is mainly expressed in pollen and is thought to contribute to the K+ conductance of the pollen tube plasma membrane [2+-binding EF hands in the cytosolic C-terminal part. TPK3 is expressed in the stromal lamellae of thylakoid membranes where it controls the transmembrane ∆pH and, thus, the light-induced proton motive force [+-selective currents observed in guard cells and mesophyll cells [+ release during stomatal closure [+ homeostasis [membrane ,37, withmembrane . The subve force . The tonll cells ,41. In g closure ,41. Thiseostasis . The bioArabidopsis. The most popular are AtHAK5 from Arabidopsis and its homologs in other species, which mediate high-affinity K+ uptake in root from very dilute soil solutions, below 10 µM (reviewed by References [+ uptake would be provided by co-transport with protons [+ uptake [+ (Rb+) uptake, and are supposed to mediate K+ efflux [+ uptake from the soil, they are also involved in K+ homeostasis, long-distance K+ transport, cell elongation, response to osmotic stress, and other unexpected functions such as adenylate cyclase activity and regulation of auxin transport [This family comprises 13 members in ferences ,84,85).  protons . KUP tra+ uptake , whereas+ efflux . KUPs ar+-selective” and “K+-permeable”, which can also transport Na+. The latter subfamily, which seems to be specific to monocots, is characterized by the presence of a conserved glycine in the selectivity filter [+ or K+ influx, or Na+/K+ co-transport [+/H+ exchangers) and CHX (cation/H+ exchangers) are cation transporters found on endomembranes including the tonoplast, and CHX transporters are also present at the plasma membrane. Some of them are involved in the control of K+ homeostasis [The HKT transporter family is divided into two subfamilies, “Nay filter . HKT traransport ,83. NHX eostasis . These fGenes encoding membrane transporters and their transcriptional regulations by climate-related stresses  are presented in + uptake is critical to reduce the effects of drought stress. K+ is taken up from a soil solution that is highly variable in composition, with K+ concentration that can range from a few µM to several mM. In Arabidopsis, below 10 µM K+ and in the absence of NH4+, only HAK5 is able to mediate K+ uptake, whereas AKT1 intervenes at higher, but still low, K+ concentrations, due to the high hyperpolarization of root cell membranes in K+-deficient soils [HAK5 expression in roots [+ absorption results in depolarization of membranes of external root cells, which promotes an unusual K+ efflux via AKT1 [+ efflux under salt stress [+ uptake from the soil at low K+ concentrations [HAK5, homologous genes in other plant species  are highly upregulated by K+ shortage; however, for at least three of them, this upregulation is suppressed by Na+ [+ uptake from the soil even in the low-K+ range [Arabidopsis  was recnditions . In paranditions , and intnditions . Neithernditions ,45. In r subunit . It imprsalinity .+ must be avoided to maintain the K+/Na+ ratio. However, Na+ entry depolarizes the plasma membrane and evokes massive loss of K+ mainly through GORK [+ efflux. In pea, they assume this function in place of GORK [Arabidopsis, KUP6 and KUP8  are alsom roots . KUP8 isORK gene . Conversin roots . The red to heat  might, trization  or in rerization . Dependi+ is transported radially (via an apoplastic or symplastic pathway) and then delivered to the xylem. KUP6 and KUP8 are expressed in the pericycle, and they enhance ABA response to inhibit lateral root formation [+ transported in the xylem sap to the shoot [SKOR gene transcript is considerably reduced in response to ABA treatment [+ currents in the maize stele [+ content in the xylem sap is accompanied by an increase in Ca2+ [+ uptake from roots, KUP7 was identified as a transporter allowing K+ efflux to the xylem especially under K+-deficient conditions , belonging to the same clade Ia as HAK5, mediate not only K+ uptake from the soil but also translocation to the xylem, possibly (at least for OsHAK5) by allowing K+ loading of xylem parenchyma cells prior to K+ efflux via SKOR-like channels [+/Na+ ratio [+ uptake and control of K+ homeostasis under salt stress [Once taken up by epidermal and cortical cells at the root periphery, Kormation . The SKOhe shoot . The SKOreatment , in accoze stele . This woze stele , since t in Ca2+  that serxylem K+ ). SystemUP7 gene . KUP7 cochannels ,98. A rechannels  highligha+ ratio . OsHAK21a+ ratio ), mainlyt stress .+ transport systems during adaptation to stress. The first transport system identified in the phloem was the Shaker channel AKT2  from Arabidopsis [KAT1 and KCO6 (AtTPK3) transcripts were also detected in isolated phloem companion cell protoplasts [KAT2 [OsHAK1 [OsHAK5 [GUS reporter gene) in phloem tissues. Due to its unique gating properties, AKT2 was extensively studied with the aim of understanding its role in the phloem transport of solutes. The AKT2 channel from Arabidopsis and its counterparts in other species substantially contribute to the phloem K+ conductance in both sieve elements and companion cells, where they have a major role in phloem K+ loading and unloading [akt2 mutant plant is not depleted in K+. Instead, it contains only half the wild-type content of sucrose [AKT2 transcript is increased in leaves in response to ABA, thus possibly favoring K+ recirculation to the root under drought stress [Due to the difficulty to access phloem sap and phloem membranes, little is known on the behaviors of phloem Kbidopsis ,99. KAT1toplasts . The KATts [KAT2 , OsHAK1  [OsHAK1 , and OsH [OsHAK5  promoternloading ,103,104. sucrose . This ist stress .+) fluxes in response to drought stress and ABA. Also, the molecular mechanisms of light-induced stomatal opening and closure were extensively studied physiological studies, guard cells became a model for the regulation of ion ferences .2+ uptake, regulated by ROS and nitric oxide [+ voltage-gated GORK channel that mediates K+ efflux [KUP6 is highly responsive to osmotic stress, especially in shoots [KUP6 gene in stress adaptation, and close their stomata more efficiently than wild-type plants in the presence of ABA [KAT1/KAT2, AKT1, GORK, and, to a much lesser extent. AKT2 and AtKC1 are expressed in guard-cell protoplasts [KAT1 and KAT2 encode inward-rectifying-forming subunits that, after tetramerization, form the KAT1 and KAT2 channels, which are the key actors of stomatal opening. Stomatal opening mechanism is an inversion of stomatal closure in terms of osmolyte accumulation and direction of fluxes. KAT1 and KAT2 mediate K+ influx that drives water influx and opens stomata [KAT1 and KAT2 genes are downregulated at the transcriptional level, as expected for genes involved in stomatal opening [GORK gene transcription, which is clearly upregulated by ABA in most tissues [+ uptake [akt1 loss-of-function mutant, the stomatal conductance is lower under drought stress and ABA has a much stronger effect on stomatal closing [ABA perception by PYR/PYL/RCAR  ABA receptors triggers an increase in cytoplasmic calcium ic oxide  that dep+ efflux ,115. Thi+ efflux ,109. In n shoots ,68. KUP6toplasts ,96,116.  stomata . Upon AB opening ,117. The tissues  was unex tissues ,73, thus+ uptake , was onl closing .+-selective TPK1 channel from Arabidopsis is essential for the control of K+ homeostasis and vacuolar K+ release during ABA-mediated stomatal closure [Although most data rely on plasma membrane channel regulation, it should be emphasized that vacuoles, which serve as solute reservoirs, play a significant role in stomatal movements. The voltage-independent, K closure ,119.+ uptake through the Arabidopsis SPIK channel [+ transport systems to the mechanisms of response to stress and their consequences on fruit quality. Most of them were obtained on grape berry, considered as a model for fleshy fruits [+ is often limiting in plants, especially under hot and dry climates, it is sometimes accumulated in excess. In grape berries, potassium accumulates after the véraison stage that marks the beginning of maturation with change in berry color and firmness, dramatic increase in sugar content, loss of acidity, and development of aromas. This K+ accumulation increased during the past decades and this was attributed to global warming, since high temperatures favor K+ accumulation in berries [+ seems to be beneficial for enhancing berry resistance to drought [+ ions, high amounts of K+ in the must are associated with high pH [+ enters in the berry mainly via the phloem. The weakly rectifying Shaker channel VvK3.1 which belongs to the AKT2 channel phylogenetic branch, would be involved in the massive K+ fluxes from the phloem cell cytosol to the berry apoplast during berry K+ loading [+ is taken up by flesh and skin cells [+ uptake into the skin cells at pre-véraison stages [+ retrieval by perivascular and flesh cells from the apoplast.Pollen germination on the stigma requires cell elongation maintained by K channel . Data ary fruits . Whereas berries . Althoug drought , it can  high pH , resulti high pH ,123. Aft high pH , K+ ente loading . Once dein cells  to be stn stages . VvK1.1,n stages . Howevern stages . In ligh+ membrane transport system activity: the SnRK2 (SNF1-related protein kinase 2) family, represented notably by OST1/SnRK2.6/SnRK2E [+ deficiency, drought, osmotic, and salt stresses [Arabidopsis genome encodes 10 SnRK2s, of which OST1/SnRK2.6 is a positive regulator of ABA-dependent stomatal movements [2+-independent signaling, whereas CIPK and most CPK kinases depend on calcium for their activity [Arabidopsis thaliana, which are combined to regulate ion homeostasis and response to ABA in a highly specific manner [A. thaliana) contain an intrinsic C-terminal calmodulin-like domain comprising EF-hand motives that directly bind calcium. When calcium levels are low, kinases are auto-inhibited, but calcium elevation triggers binding to EF-hands and conformational changes that relieve the auto-inhibition [+ channel regulation, as described below.Three main kinase families share the regulation of K6/SnRK2E , the SnR6/SnRK2E ), and th6/SnRK2E   and its counterparts in other species. In whole rice seedlings, OsCIPK23 is induced by ABA [TaCIPK23 is induced by drought, salt, and ABA [GmCIPK20 (closest homolog of AtCIPK23) is induced by drought stress in leaves and stems but not in roots [CIPK6 were also identified in other species. BnCIPK6 and BnCBL1 genes (from Brassica napus) are both upregulated by NaCl, mannitol, and ABA in roots, in accordance with the increased salt tolerance of transgenic Arabidopsis plants overexpressing these genes [The first kinase to be clearly identified as a partner of a Kthaliana ,154. Mut+ medium . CIPK23 + medium . To actis or ABA , with thficiency , it is n drought ,160, and drought . Root-sp drought . The CBLng zones . CIPK23 ng zones ,154 and ng zones . Conversng zones . AKT1 alng zones ,140, whong zones , and witng zones ,140. TheK23/CBL1 , which a+ uptake , and TaC+ uptake ,188. Trad by ABA , and, inin roots . Homologse genes .+ uptake in low-K medium [Arabidopsis CIPK23/CBL1 complex in yeast [+ uptake by CIPK/CBL complexes at the root/soil interface.Interestingly, HAK5 is the target of the same CIPK23/CBL1 complex as AKT1. CIPK23 phosphorylates HAK5, resulting in activation of the transporter, required for KK medium . This rein yeast . Figure + efflux is mediated by GORK at the plasma membrane. This efflux is reduced in 14-3-3 mutants. Indeed, salt stress is expected to trigger membrane depolarization and a rise in cytosolic calcium that activates CPKs together with 14-3-3s, to promote GORK phosphorylation leading to its activation [Recently, CPK21 was identified as a kinase activated by 14-3-3 proteins, phosphorylating the GORK C-terminal cytoplasmic domain. Following a sudden salt stress, a transient Ktivation .From the beginning of its characterization, AKT2 received most of the attention due its unique gating properties, which allow it to combine a time-dependent voltage-activated current and an instantaneous ohmic current with the ability to switch between these two gating modes . The mecAtPP2CA is a negative regulator of ABA signaling ,178 sele+-ATPase and Kin channels  protein kinases from Vicia faba [A. thaliana, homologous to AAPK, was identified as a key player of ABA-mediated stomatal closure, acting upstream of ROS production [ost1 mutants, suggesting that OST1 inhibits KAT1 [Xenopus oocytes is not inactivated by OST1 [+ transport activity, thus, await further investigation.During ABA-mediated stomatal closure, mechanisms leading to blue-light-induced stomatal opening are inhibited. This includes inactivation of Heference  and refecia faba ,200. Thecia faba , stronglcia faba . Later ooduction . The ABAits KAT1 . OST1 phits KAT1 , and dirits KAT1  , CPK3, 4, 6, 7, 11, 13, and 33 were pinpointed as being expressed in guard cells . A systecurrents   remains to be clarified. Based on mutant phenotypes, it appears that its activating partners CIPK23 and CBL1/CBL9 also counteract stomatal closure in the presence of ABA and increase sensitivity to drought [CIPK23, the CIPK6 gene, encoding another activating partner of AKT1 [+ influx [AKT1 is activated by the CIPK23/CBL1 complex in guard cells as it is in roots  Figure . It is t drought . AKT1 al drought , an ABA- drought , which i drought ,140. AIP drought , interac drought . Togethe of AKT1 . Its ove of AKT1 . The CIP+ influx ,165,193.GORK transcripts in response to ABA [Xenopus oocytes is inhibited by CPK3, CPK7, and CPK13, and it is activated by CPK6 and CPK33 [cpk33 mutants do not close their stomata in the presence of calcium. However, they are also more resistant to drought and their stomata close more efficiently in the presence of ABA [+, suggests the existence of additional post-translational regulatory mechanisms [+ efflux from guard cells, is phosphorylated by OST1 [Xenopus oocytes [As the main channel mediating Kout currents at the plasma membrane of guard cells, GORK is expected to be a target of regulatory mechanisms to control stomatal closure including the closing speed. However, its regulatory network is much less characterized than that of AKT1 or KAT1. Unlike other cell types, guard cells do not induce e to ABA ,73 and, nd CPK33 . Accordie of ABA . GORK ace of ABA , thus prchanisms . KUP6, a by OST1 , while t oocytes .+ fluxes at the plasma membrane, K+ loading and unloading of vacuoles play a central role in the control of guard-cell turgor. The TPK1 tonoplast channel seems to be the main K+-selective channel involved in that process. It is phosphorylated by CPK3 that is highly expressed in guard cells [Although much less studied than Krd cells  and resprd cells . In resprd cells .VvCIPK04 gene, encoding a kinase strongly activating VvK1.2, is upregulated by drought stress in the ripening berry [+ channels, interact with each other in a specific manner and corresponding genes are differentially regulated [+ berry content correlated to temperature increases can be attributed at least in part to Shaker channels in relation to SnRK2 and CIPK kinases.The AKT1-related VvK1.1, VvK1.2, and AKT2-related VvK3.1 genes are strongly upregulated by drought stress , and these channels are activated by CIPK/CBL couples ,51,59. Gegulated . Among tegulated . In the egulated  and ABA egulated ,210. It + homeostasis is crucial. In the case of salt stress, Na+ is accumulated in vacuoles, and K+ is released from the vacuole to maintain the cytosolic K/Na ratio. For efficient sequestration of Na+ in the vacuoles, slow and fast vacuolar currents allowing Na+ leakage from the vacuole to the cytosol must be inactivated [+ flux back to the cytosol to counterbalance Na+ entry into the vacuole and compensates for K+ efflux through GORK at the plasma membrane [PeTPK1 results in improvement of salt tolerance [Arabidopsis salt-stressed plants, but not those of control plants, in vitro phosphorylate the TPK1 N-terminus [As highlighted above, maintaining Kctivated . Under smembrane . In poplolerance . Leaf exterminus . Lookingterminus  identifiterminus  , a syntaxin  associated with the plasma membrane. This protein accumulates in leaves after ABA treatment [+ channels [Arabidopsis homolog of Nt-SYR1) modify KAT1 distribution and mobility at the plasma membrane [+ channels is the voltage sensor, and binding is favored by hyperpolarization, thereby allowing coordination of vesicle trafficking for cell volume adjustment and K+ channel voltage-dependent activation [Xenopus oocytes. Altogether, SYP121 and VAMP have complementary actions on K+ channel activities [As discussed above for AKT2, an increase in channel activity at the plasma membrane can result from a change in channel gating properties  or from a regulation of membrane targeting. Screening for guard-cell tobacco RNAs able to induce ABA-dependent Cln et al.  isolatedreatment . A cleavchannels . Visualimembrane . During membrane ), KAT1 imembrane . SYP121 membrane . The bintivation . Other Stivities   can be involved in the regulation of both K+ and mineral anion transport. The main mineral anions in plants are Cl− and NO3−. Anion channels of the SLAC1/SLAH (slow anion channel 1/ SLAC1 homolog) family (five members in Arabidopsis) mediate voltage-independent slow and massive chloride and nitrate fluxes at the plasma membrane, for root anion uptake, transfer of anions to the shoot, and stomatal closure [As the major cation in plant cells, K closure . Recent  closure .+ and anion effluxes and modulation of the speed of ABA-triggered stomatal closure. SLAC1 interacts with and is activated by the OST1 kinase, whose activity is inhibited by AtPP2CA and its close relative ABI1 (ABA-insensitive 1) in an ABA-dependent manner [In guard cells, SLAC1 and SLAH3 mediate nitrate and chloride transport. SLAC1 was found inhibited by AtPP2CA , like AKt manner ,144.cpk3 cpk6 double knock-out mutant, Ca2+- and ABA-activated S-type anion channel currents are abolished, and stomatal closure is partially impaired [Xenopus oocytes and to phosphorylate the SLAC1 N-terminus at a much higher speed than CPK23 and OST1 [+ channel AKT1 and the high-affinity transporter HAK5, also interacts with and activates SLAC1 and SLAH3. Like CPK23, it phosphorylates the N-terminus of SLAC1 in a calcium-dependent manner, but there exist specificities for target phosphorylation sites [Like OST1, kinases of the CPK family, activated by calcium, also interact with SLAC1 and SLAH3 to modulate anion efflux in guard cells. CPK21 and CPK23 both activate SLAC1 and SLAH3 in an ABA-and ABI1-dependent manner, but with contrasting effects on channel currents and calcium sensitivity ,139,141.impaired . Accordiand OST1 . CPK21 [and OST1 . Finallyon sites .− uptake [− uptake under high external nitrate in Xenopus oocytes [− uptake, CHL1 is also involved in stomatal opening and plant drought susceptibility [In the nitrate transporter family, CHL1 (NRT1.1/NPF6.3) , involved in root NO3− uptake , is also− uptake . CBL1 al oocytes . This in oocytes . In additibility . CIPK23 tibility . Since tXenopus oocytes. AtPP2CA [Clade A PP2C phosphatases are at the center of ABA signaling pathways. Their incorporation into kinase/channel complexes most often results in complete abolition of the effect of the kinases. Indeed, AtPP2CA, ABI1, and/or ABI2 drastically inhibit the effect of OST1 ,144,167, AtPP2CA ,138 and  AtPP2CA , despite AtPP2CA . In para AtPP2CA , GORK [1 AtPP2CA , and SLA AtPP2CA  activiti AtPP2CA . Thus, P AtPP2CA ,136,144  AtPP2CA  via bind AtPP2CA  or depho AtPP2CA .cipk23 mutants  rather t stress , but alsnel GORK . CPK33 anel GORK  but downnel GORK . The cpkmannitol , while Cmannitol , which wmannitol  and CPK2mannitol . Clade Amannitol . Dependi


Our purpose was to combine intravoxel incoherent motion diffusion-weighted MR imaging (IVIM-DWI) and magnetic resonance spectroscopy (MRS) to differentiate osteoporotic fractures from osteolytic metastatic vertebral compression fractures (VCFs).A total of 70 patients with VCFs were included and divided into two groups, according to their causes of fractures based on pathological findings or clinical follow-up. All patients underwent conventional sagittal T1WI, T2WI, STIR, IVIM-DWI, and single-voxel MRS. The diffusion coefficient (D), pseudo diffusion (D*), and perfusion fraction (f) parameters from IVIM-DWI and the lipid water ratio (LWR) and fat fraction (FF) parameters from MRS were obtained and compared among groups. Furthermore, the diagnostic performance of MRS, IVIM-DWI, and IVIM-DWI combined with MRS for differentiation between osteoporotic and osteolytic metastatic VCFs was assessed by using receiver operating characteristic (ROC) curve analysis.D* . The area under the receiver operating characteristic (ROC) curve of MRS, IVIM-DWI, and IVIM-DWI combined with MRS were 0.73, 0.88, and 0.94, respectively. Among these, the IVIM-DWI combined with MRS showed the highest sensitivity, specificity, and accuracy, which are 90.63% (29/32), 97.37 % (37/38), and 94.29% (66/70), respectively.Compared with the osteoporotic group, the metastatic group had significantly lower values for f, D, and FF, but higher IVIM-DWI combined with MRS can be more accurate and efficient for differentiation between osteoporotic and osteolytic metastatic VCFs than single MRS or IVIM-DWI. Vertebral compression fractures (VCFs) are common and occurred frequently among the elderly, which are caused by osteoporosis. However, the spine is also a frequent location of metastatic that may result in pathologic fractures in one third of patients with cancer. Regular diagnosis is very important for the option of treatments and prognosis for patients; moreover, the unnecessary vertebral biopsy can be evitable in patients with osteoporotic VCFs . Though Diffusion-weighted imaging (DWI) has proven to be useful in oncological imaging for tumor detection and characterization –5. HowevIn this study, our purpose was to examine whether IVIM-DWI combined with MRS as a noninvasive method can be used to differentiate osteoporotic fractures from metastatic VCFs.n = 138) in the orthopedics suffered from VCFs and underwent CT examination without definite diagnoses and were recruited in this study between August 2016 and July 2017. Then, all participants underwent quantitative CT bone densitometry examination and received 3.0-T spinal MRI, including conventional sagittal T1WI, T2WI, STIR, and IVIM diffusion-weighted imaging and 1H-MRS. Inclusion criteria for all patients were (i) an acute or subacute vertebral fractures for less than 3 months, (ii) bone marrow edema at the fracture sites, (iii) aged 18 years or older, (iv) no MRI contraindications , and (v) osteoporosis (with a T score below 2.5). Among the 138 patients, the following exclusion criteria were applied: (i) Unsatisfactory image quality or an artifact caused by a metal device (n = 5), (ii) old compression fractures (n = 9), (iii) chemotherapy or radiation therapy before MRI examination (n = 11), (iv) patients with presumed osteoporotic or metastatic without more than 3 months follow-up or did not have pathological confirmation (n = 10), and (v) primary vertebral tumors  and multiple myeloma (n = 3)), sclerotic metastases (n = 6), diffuse hematologic disorders (n = 2), spondylitis (n = 2), spinal tuberculosis (n = 5). Therefore, a total of 70 patients with VCFs were included, and the onset time for the fractures patients suffered from was ranged from 6 h to 15 days before MR imaging , (2) no primary tumor, (3) follow-up CT (n = 17), and/or MRI (n = 19) for more than 3 months: edema disappeared, non-morphologic signs of malignancy , as well as the disappearance of clinical pain.The osteoporotic group consisted of 38 patients with osteoporotic VCFs, including 2 cervical vertebrae, 20 thoracic vertebrae, and 16 lumbar vertebrae. The diagnosis criteria and distributions for the osteoporotic fracture were (1) surgery and histopathologic examination (n = 6), thoracic vertebra (n = 16), and lumbar vertebra (n = 10). The sources of the primary neoplasms were lung cancer (n = 10), breast cancer (n = 7), prostate cancer (n = 6), renal cell carcinoma (n = 3), colorectal cancer (n = 2), ovarian cancer (n = 2), pancreatic cancer (n = 1), and thyroid carcinoma (n = 1). The diagnosis criteria and distributions for the metastatic fracture were (1) primary malignant tumor in other sites (n = 32), (2) CT-guided biopsy (n = 3), (3) follow-up CT (n = 12), and/or MRI (n = 17) for more than 3 months: the persistent of bone marrow edema, morphologic signs of malignancy , and the persistent of clinical pain.The metastatic group consisted of 32 patients with malignant VCFs, including cervical vertebra  with a quadrature spine surface coil. The routine MRI sequences were obtained firstly including T1-weighted (echo time (TE) = 10 ms, repetition time (TR) = 550 ms) and T2-weighted . The STIR  images of 18 sagittal slices with a slice thickness of 4 mm were acquired (field of view (FOV) = 256 mm × 256 mm, matrix size = 256 × 256).b values = 0, 50, 100, 150, 200, 400, 600, and 800 s/mm2, respectively. The mean acquisition time of the IVIM sequence was 6:54 min.The IVIM-DWI sequence was performed with the following parameters: TR = 1600 ms, TE = 72 ms, slice thickness = 5 mm, FOV = 340 mm × 340 mm, matrix size = 256 × 256, and 3.Similarly, MRS was performed using the point resolved spectroscopy (PRESS) sequence with the following parameters: TR = 2000 ms, TE = 30 ms, NEX = 64, bandwidth = 2000 Hz, flip angle = 90°, without water suppression, and total scan time of 4:27 min. The dimensions of voxels were varied according to the configuration of the fracture. The MRS voxels were positioned centrally within the STIR hyperintensity of the fractured vertebrae, avoiding the cystic degeneration and necrosis. On average, the voxels were comparable at 3.0 ± 0.5 cmAll MR images were retrospectively identified by two independent radiologists , who were blinded to the purpose of this study. For each patient, the acquired MRS images were pre-processed by applying the Siemens Syngo.via workstation. The LC-Model program was designed to determine the relative peak areas of the signals of water at 4.7 ppm and lipid at 1.3 ppm. Both parameters of lipid water ratio (LWR) and fat fractions (FF) were calculated according to the following equations:Slipid is the area under the lipid peak, and Swater is the area under the water peak.where www.mitk.org, an open-source software), which yielded values for D, D* and f. Regions of interest  were manually drawn independently by the two radiologists on each lesion which exhibited the maximal lesion area on IVIM parameter maps corresponding to the MRS voxel directly.The IVIM-derived parameters for each patient were estimated with MITK Diffusion  analysis, which calculated the value of the sensitivity, specificity, and diagnostic accuracy. Furthermore, the areas under the ROC curve (AUC) were compared for significant difference between the MRS, IVIM-DWI, and IVIM-DWI combined with MRS.Firstly, inter-observer agreements were evaluated by calculating intraclass correlation coefficients (ICCs). The value P > 0.05) and gender (P > 0.05) between two groups. All demographic characteristics for patients were presented in Table The osteoporotic group consisted of 38 patients with osteoporotic VCFs , and there were 32 patients with malignant VCFs in the metastatic group . There were no differences in age , the ROC of MRS for differentiation between osteoporotic and osteolytic metastatic VCFs was drawn by FF. The sensitivity, specificity, and accuracy of MRS in differentiating osteoporotic from osteolytic metastatic VCFs were 87.50% (28/32), 57.89 (22/38), and 71.43% (50/70), respectively , the sensitivity, specificity, and accuracy of IVIM-DWI in differentiating osteoporotic from osteolytic metastatic VCFs were 78.13% (25/32), 89.47 (34/38), and 84.28 (59/70), respectively , the diagnostic accuracy was 94.29 (66/70). The diagnostic sensitivity and specificity were 90.63% (29/32) and 97.37 (37/38), respectively  , 0.88 (95% CI 0.772–0.941), and 0.94 (95% CI 0.88–0.994), respectively , which suggested that the diagnostic performance was significantly improved. Among 38 osteoporotic fractures, 16 cases were diagnosed as metastatic by MRS, and 12 cases were diagnosed as osteoporotic with a combination of IVIM-DWI and MRS, suggesting that IVIM-DWI could differentiate the nature of vertebral fracture and provided valuable information according to the viewpoint of restricted diffusion of water molecules. Among 32 metastatic fractures, 4 cases were diagnosed as osteoporotic fracture by IVIM, and 3 cases were diagnosed as malignant by IVIM-DWI combined with MRS, because the primary tumors were prostate cancer and small cell lung cancers, and the changes in cell density of these two types of metastatic diseases were not obviously significant. It was easy to misdiagnose the nature of vertebral fracture which only based on multi-There were several limitations to our study. Firstly, metastatic vertebral fractures originate from different types of primary tumors and are not grouped according to the primary malignancy, which may lead to different perfusion patterns and signal differences. Secondly, there were different diffusions between osteogenic and osteolytic metastasis; therefore, the study excluded osteogenic metastases, and the group of metastatic vertebral fractures are mostly osteolytic metastasis; osteogenic metastasis could be incorporated into the study as one of the research directions in the future. Thirdly, the majority of diagnoses were based on clinical and radiographic evidence that biopsy confirms that the nature of the fractures was not routinely performed. Finally, other benign and malignant lesions, such as spondylitis (suppurative or tuberculous), primary vertebrae body tumors, and multiple myeloma were not included in this study.Overall, IVIM-DWI combined with MRS can improve the differential diagnostic performance of osteoporotic and osteolytic metastatic vertebral fractures, which is of great significance to guide the clinical development of the correct treatment plan.


Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by excessive inflammatory and immune responses and tissue damage. Increasing evidence has demonstrated the important role of programmed cell death in SLE pathogenesis. When apoptosis encounters with defective clearance, accumulated apoptotic cells lead to secondary necrosis. Different forms of lytic cell death, including secondary necrosis after apoptosis, NETosis, necroptosis, and pyroptosis, contribute to the release of damage-associated molecular patterns (DAMPs) and autoantigens, resulting in triggering immunity and tissue damage in SLE. However, the role of autophagy in SLE pathogenesis is in dispute. This review briefly discusses different forms of programmed cell death pathways and lay particular emphasis on inflammatory cell death pathways such as NETosis, pyroptosis, and necroptosis and their roles in the inflammatory and immune responses in SLE. Systemic lupus erythematosus (SLE) is a highly heterogeneous autoimmune disease that affects almost all organs and tissues . It is cα, and TRAIL, while the intrinsic pathway is triggered by DNA damage, endoplasmic reticulum stress, cytokine withdrawal, or lack of nutrient support. Both apoptotic pathways require activation of caspase family members, caspase-8 and caspase-9 for the extrinsic pathway and the intrinsic pathway, respectively. Ultimately, the pro-caspase-3 is cleaved into caspase-3 and activated, resulting in the cleavage of the cellular substrates and eventual apoptosis [β and IL-10 are released during the phagocytosis of apoptotic cells [In 2008, we proposed a cell death recognition model for the immune system that the consequences of immune responses, tolerance or adaptive immune responses, are dependent on the ways of cell death . Indeed,poptosis . Apoptotpoptosis . In addiic cells . And impic cells . In a moic cells . Therefoic cells . Loss ofic cells .+ T cells from SLE patients through the Fas-caspase axis [Glomerular apoptotic nucleosomes were targeted by anti-dsDNA autoantibodies in human lupus nephritis . Apoptotase axis . Apoptotase axis . In addiase axis , 25. Lupase axis . Consistase axis . Similarase axis , 29. In Apoptotic cells must be engulfed efficiently by phagocytes to prevent the release of cell ingredients that may activate the immune system. However, impaired clearance of apoptotic cells in SLE is thought to disrupt the balance of the immune system. Efficient clearance of apoptotic cells mainly involves the recognition and engulfment by professional phagocytes. Indeed, apoptotic cell receptors and bridging molecules related to the recognition and engulfment have been found to be defective in SLE. Tyro-3, Axl, and Mer (TAM) receptor protein tyrosine kinases are important receptors on phagocytes for the clearance of apoptotic cells by their recognition of ligands that are bound to PS exposed on the membrane of apoptotic cells , 31. Merα in plasmacytoid dendritic cells (pDCs) [Owing to the impaired clearance of apoptotic cells, accumulated apoptotic cells may undergo secondary necrosis by which cellular components are released. Necrosis is characterized by loss of plasma membrane integrity, exposure of autoantigens, and release of DAMPs and therefore induces autoimmunity. Autoantibodies promoted the uptake of secondarily necrotic cell-derived material by phagocytes, accompanied by secretion of huge amounts of inflammatory cytokines . Furthers (pDCs) . Collectβ2 integrin in a ROS-independent manner [A hallmark feature of SLE is the presence of antibodies against various nuclear antigens, especially anti-double-stranded (ds) DNA antibodies. NETosis, a specialized cell death form in neutrophil, is considered as a major source of modified and/or externalized autoantigens in SLE . In partt manner . As in ct manner –57. Recet manner , 59.β and IL-18, further exacerbating the inflammatory responses. In turn, IL-18 can induce NETosis in human neutrophils, creating a proinflammatory feed-forward loop that may result in disease flares [α and activates the immune system [NETosis leads to the exposure of autoantigens to the immune system and the release of DAMPs to activate the immune responses. Native and oxidized DNA bound to NETs can activate pDCs to produce higher levels of IFN in a Toll-like receptor 9-dependent or a STING-dependent manner, respectively , 60, 61.e flares . An addie flares . Furthere flares . NETs mae flares . Tissue e flares . And thre system . Based oα, IL-8, and IL-6 [SLE patients display a distinct subset of proinflammatory neutrophils, named low-density granulocytes (LDGs), in the peripheral blood mononuclear cell (PBMC) fraction . LDGs shand IL-6 . Functioand IL-6 , 71. In and IL-6 , 72, 73.and IL-6 . Meanwhiand IL-6 . On the and IL-6 , 75. On and IL-6 . The linand IL-6 . Thus, eand IL-6 .Some animal studies provided further evidence for the role of NETosis in the pathogenesis and development of SLE. Inhibition of peptidylarginine deiminase blocked NETosis and protected against lupus-related damage to the vasculature, kidneys, and skin in various lupus-prone mouse models , 79. MRLβ and IL-18 [+ T cells, hepatocytes, vascular endothelial cells (VECs), tubular epithelial cells, and many other cell types [β and pro-IL-18 into mature inflammatory cytokines IL-1β and IL-18, respectively. On the other hand, activated caspase-1 directly cleaves gasdermin D (GSDMD) and releases active N-terminus subunit that binds to phosphoinositides in the plasma membrane and forms pore (about 10-14 nM in size). The pore formation results in the loss of osmotic potential, cytoplasmic swelling, release of inflammatory factors, and finally cell explosion. In the noncanonical pathway, caspase-4,5 or caspase-11 in the host cytoplasm can directly recognize lipopolysaccharide (LPS) from gram-negative bacterial and then cleave GSDMD, leading to host cell pyroptosis [Pyroptosis is a lytic and inflammatory form of programmed cell death induced by a variety of danger signals. It is characterized by gasdermin family-mediated pore formation on the plasma membrane, cell swelling, and eventual lysis, followed by release of cellular contents, especially inflammatory mediators IL-1nd IL-18 . Althougnd IL-18 , it can ll types –89. To dll types . The actroptosis . More reroptosis . This imβ and IL-18, the most important inflammatory cytokines released by pyroptotic cells, can trigger a secondary inflammatory response in neighboring cells. IL-1β can activate the NF-κB pathway through the IL-1 receptor, leading to the generation of inflammatory cytokines including cyclooxygenase-2 (COX-2) and IFN-γ [α, IL-6, and IL-8 primarily via the MAPK p38 pathway [Numerous studies have suggested that pyroptosis can potentiate the inflammatory reaction and enhance adaptive immune responses by the release of various cellular contents. IL-1nd IFN-γ . Meanwhi pathway . In addi pathway . Importa pathway , 95. In  pathway , 97. The pathway , 99. In  pathway . In the  pathway .β. Indeed, suppression of P2X7 receptor by its selective antagonist brilliant blue G reduced the severity of nephritis and improved the survival of MRL/lpr mice by inhibiting the NLRP3 inflammation activation and decreasing the production of proinflammatory cytokines [Increasing studies strongly suggest the important role of pyroptosis in the pathogenesis and progression of SLE. NLRP3 inflammasome, one of the inflammasome sensors mediating pyroptosis, was found hyperactivated in patients with SLE and lupus nephritis (LN) , 102. Inytokines , 107. Inytokines . The expytokines . In addiytokines . Elevateytokines , 112. Fuytokines , 114. Anytokines . Indeed,ytokines , 117. Reytokines . Importaytokines . Interesytokines . Surprisytokines . The finNecroptosis is a caspase-independent form of programmed necrotic cell death characterized by receptor-interacting protein kinase 3- (RIPK3-) mediated phosphorylation of mixed lineage kinase domain-like protein (MLKL) . It is tβ inflammatory responses [β release [β [α-induced necroptosis can be engulfed by human macrophages and dendritic cells, leading to the secretion of macrophage cytokines and maturation of dendritic cell [α belong to the IL-1 family and have the potential to induce inflammatory responses and to amplify immune responses [α and IL-33 were found in the serum of RIPK1-deficient mice, but dependent on the presence of RIPK3 and MLKL [γ) [α, IL-1β, and IFN-γ) [α and IL-8 by targeting MLKL [Certain evidence has demonstrated that necroptotic signaling could also induce the NLRP3 inflammasome activation and eventual pyroptosis, further amplifying the inflammatory response. RIPK3 that is essential for necroptosis can promote the NLRP3 inflammasome activation and IL-1esponses . Similar release , 128. Fulease [β , 129. Intic cell . Both ILesponses . High leand MLKL . Furtherand MLKL . In vivoMLKL [γ) . In addid IFN-γ) . MicroRNing MLKL . Similaring MLKL . Necropt“Natural” necroptosis mostly occurs in infectious pathological conditions. Recently, some studies have suggested that necroptosis may be implicated in the pathogenesis and development of SLE. Importantly, necroptosis was observed in B cells from SLE patients . The finAutophagy is a highly conserved lysosome-mediated catabolic and homeostatic process for digesting unnecessary or dysfunctional cellular organelles and recycling nutrients . Based oγ-producing T cells from SLE patients, and the percentage of autophagy positively correlated with the disease activity [+ T cells [α by pDCs [β production via targeting TANK-binding kinase 1 [α production in Tlr7.1 tg mice [α from patients with LN could induce autophagy in both murine and human podocytes. With regard to intervention, autophagy inhibitors exacerbated podocyte damage while its inducer relieved the injury. Consistently, the autophagy inducer, rapamycin, decreased disease activity and reduced prednisone requirement in refractory SLE patients in clinical practice [Increasing studies focus on the link between autophagy and autoimmune diseases, especially SLE. Genome-wide association studies (GWAS) have identified that several autophagy-related genes are correlated with susceptibility of SLE, including ATG5, CDKN1B, DRAM1, CLEC16A, and ATG16L2 . In furtactivity . Moreove T cells . Autopha T cells . Autopha by pDCs . MicroRNkinase 1 . Loss of tg mice . Importa tg mice , 160. Th tg mice . Interes tg mice , 161. Co tg mice . Repeate tg mice . Interes tg mice , 164. Inpractice , 166. Sipractice .A large number of researches have provided strong evidence for the concept that dysregulation in cell death pathways and defective clearance of death materials trigger autoimmunity and contribute to the pathogenesis and progression of SLE. In SLE, accelerated cell death occurs and dying cells cannot be cleared promptly and effectively, leading to the exposure of nuclear and cytoplasmic autoantigens and release of DAMPs that work together to induce autoimmune responses and inflammatory responses. Lytic and inflammatory cell death, including secondary necrosis after apoptosis, NETosis, necroptosis, and pyroptosis, plays important roles in SLE pathogenesis and progression. However, owing to the lack of markers of necroptosis, human disease processes that involve necroptosis in vivo are hard to investigate . Thus, f


Capparis spinosa L. (C. spinosa) has been used as food and traditional medicine and shows anti-inflammatory and anti-oxidant activities. Here, we prepared the C. spinosa fruit ethanol extracts (CSEs) using different procedures and investigated the effects of CSE on the maturation of mouse bone marrow-derived dendritic cells (DCs) in the absence or presence of lipopolysaccharide (LPS). DC maturation and cytokine production were detected by flow cytometry and ELISA, respectively. We obtained three different CSEs and dissolved in water or DMSO, named CSE2W, CSEMW, CSE3W, CSE2D, CSEMD, and CSE3D, respectively. These CSEs showed different effects on DC maturation. CSEMW and CSEMD significantly increased the expressions of CD40, CD80, and CD86, in a dose-dependent manner. CSE2W and CSE2D also showed a modest effect on DC maturation, which enhanced the expression of CD40. CSE3W and CSE3D did not change DC maturation but suppressed LPS-induced DC maturation characterized by the decreased levels of CD40 and CD80. CSE3W and CSE3D also significantly inhibited the secretions of IL-12p40, IL-6, IL-1β, and TNF-α induced by LPS. CSE3W further increased the level of IL-10 induced by LPS. Moreover, CSE3D suppressed LPS-induced DC maturation in vivo, which decreased the expressions of CD40 and CD80. These results suggested that CSE3W and CSE3D might be used to treat inflammatory diseases. Capparis spinosa L. is a kind of TCM and has been used to treat gastro-intestinal problems, rheumatism, gout, diabetes, inflammation, kidney, and liver diseases [C. spinosa  mainly distributed in Xinjiang Uygur Autonomous Region, which is a kind of sand binding plant and its buds, leaves, roots, and fruits have been used in traditional Uighur Medicine to treat gout and rheumatoid arthritis for a long time [C. spinosa contains a lot of biochemical compounds including flavonoids, alkaloids, polyphenols, saponins, terpenoids, lectin, essential oils, glycosinolate, and glycosides [C. spinosa on the maturation and cytokine production of DCs is still elusive.Traditional Chinese medicines (TCM) have been used to treat various kinds of diseases for a long history. Accumulating evidence has shown that TCM exerts immunoregulatory effects to prevent or treat infection or autoimmune diseases ,2. TCM cdiseases ,5,6,7. Iong time . Recentlycosides ,10,11,12ycosides ,14,15,16ycosides ,18, antiycosides , anti-arycosides , anti-tuycosides ,17,21, aycosides ,24,25,26ycosides ,28, antiycosides , anti-heycosides , anti-hyycosides , hypolipycosides , and immycosides . However+ helper T (Th) cell subsets that helped CD8+ T cells to generate cytotoxic T lymphocytes and B cells to produce antibodies [C. spinosa can affect the DC activation status to exert its some pharmacological effects, such as anti-inflammatory, anti-allergic, and immunomodulatory, we prepared C. spinosa fruit ethanol extracts (CSEs) and detected their effects on the maturation and cytokine production of DCs, especially in the presence of lipopolysaccharide (LPS).DCs are professional antigen presenting cells and play a pivotal role in the immune system, which link the innate and adaptive immune responses. The activation status of DCs, including maturation and cytokine secretion, determined the development of CD4tibodies ,35. TherCSEs were prepared using ethanol extract following the extraction with petroleum ether and ethyl acetate according to previous description . To sepa+Annexin V−) and apoptotic (PI−Annexin V+ and PI+Annexin V+) DCs  or CSE2D, CSEMD, and CSE3D according to their flavonoid contents  were used to treat DCs. After 12 h, DCs were collected and stained with PI/Annexin V kit. Samples were analyzed by flow cytometry. The results showed that all CSEs in selected concentrations did not significantly change the frequencies of necrotic (PI V+) DCs , suggestSeveral studies including ours have been reported that polysaccharides from herbal medicines can promote DC maturation and cytokine production ,40,41,42C. spinosa have anti-inflammatory activity [It has been shown that flavonoids in activity ,16. TherThe above results showed that both CSE3D and CSE3W did not promote DC maturation and cytokine production. We wondered whether CSE3D and CSE3W could suppress DC maturation induced by LPS. DCs were treated with different flavonoid concentrations  of CSE3D and CSE3W in the presence of LPS. After 12 h, the stained samples were analyzed by flow cytometry. The results showed that both CSE3D and CSE3W significantly suppressed the expression of CD40 induced by LPS, in a dose-dependent manner. CSE3D also significantly suppressed the expression of CD80 induced by LPS but not for CSE3W A.The levels of anti-inflammatory IL-10 and pro-inflammatory cytokines including IL-12p40, IL-6, IL-1β, and TNF-α in supernatants of the above samples were determined by ELISA. We found that the levels of all these pro-inflammatory cytokines induced by LPS were significantly decreased by CSE3D and CSE3W B. Moreov+) by flow cytometry. The results showed that LPS significantly increased the expressions of CD40 and CD80 but CSE3D did not significantly change the expressions of all these molecules on DCs in vivo  of CSE3D by footpads. Mice injected with LPS alone were used as a positive control. After 24 h, popliteal lymph nodes were isolated and lymphocytes were used to analyze the expressions of CD40, CD80, CD86, and MHC II on DCs  of CSE3D together with 100 ng/mouse of LPS by footpads. After 24 h, lymphocytes in popliteal lymph nodes were isolated to analyze DC maturation by flow cytometry. We observed that 1.6 and 0.8 μg/mouse of CSE3D significantly inhibited the expressions of CD40 and CD80 induced by LPS, respectively B. The exC. spinosa using different procedures and detected the effect of these extracts on DC maturation both in vitro and in vivo. We found that CSE2W, CSE2D, CSEMW, and CSEMD could promote DC maturation but CSE3W and CSE3D did not promote DC maturation. Interestingly, CSE3W and CSE3D suppressed DC maturation and pro-inflammatory cytokine production induced by LPS in vitro. CSE3D also suppressed DC maturation in vivo.We prepared the ethanol extracts of C. spinosa contained lots of components and showed various pharmacological activities including anti-inflammatory [C. spinosa are contributed to the anti-inflammatory activity [It has been reported that ammatory . Severalactivity ,16. DCs activity ,44. HereSome polysaccharides of herbal medicine could promote DC maturation and cytokine production ,40,41,42Taken together, CSE3 could suppress DC maturation and cytokine production induced by LPS both in vitro and in vivo, suggesting that CSE3 might be used as anti-inflammatory agent to treat arthritic and allergic diseases.C. spinosa ethanol extracts were prepared using the procedures that were shown in C. spinosa was crushed into powder. 400 g of powder was extracted using 2.4 L of absolute ethanol at 60 °C three times (2 h/time). The extracts were collected, filtrated, and concentrated by a rotary evaporator. For CSE3 and CSEM, the ethanol extract was concentrated to 100 mL, 400 mL of distilled water was added and then extracted with petroleum ether three times. The extract was divided into three layers when it was extracted with petroleum ether. On the one hand, the middle layer was isolated and lyophilized and named as CSEM. On the other hand, the extract was treated with NaCl until it was divided into two layers. The water fraction was isolated and extracted with ethyl acetate three times, then the water fraction was concentrated by a rotary evaporator. During evaporation, crystallized NaCl was timely removed. Finally, this fraction was lyophilized and named as CSE3. For CSE2, the ethanol extract was concentrated to ethanol-free, 400 mL of distilled water was added and then extracted with petroleum ether three times. The extract was divided into two layers. The water fraction was isolated and extracted with ethyl acetate three times, then the water fraction was lyophilized and named CSE2. These CSEs were dissolved in distilled water or DMSO  at 200 mg/mL and named CSE2W, CSEMW, and CSE3W; or CSE2D, CSEMD, and CSE3D, respectively.The The contents of flavonoids and polysaccharides were determined by AlCl3-KAC and anthrone-sulfuric acid method, respectively.BALB/c mice were, aged 6–8 weeks, purchased from the Beijing laboratory animal research center . Mice were housed in a temperature-controlled, light-cycled animal facility of Xinjiang University. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Xinjiang Key Laboratory of Biological Resources and Genetic Engineering and carried out under the guidelines of the Animal Care and Use Committee of College of Life Science and Technology, Xinjiang University.Mice (3 mice/group) were injected with different concentrations  of flavonoids in CSE3D or LPS (100 ng/mouse) by both sides of footpads (25 μL/footpad). After 24 h, popliteal lymph nodes were isolated to analyze DC maturation by flow cytometry.Immature DCs were induced from bone marrow cells of BALB/c mice in the presence of 20 ng/mL of GM-CSF (Peprotech) according to the previous protocol . These cUpon CSEs or LPS treatment, the levels of IL-12p40, IL-10, IL-6, IL-1β, and TNF-α in supernatants of the above samples were measured by ELISA kit according to the manufacturer’s instruction . The OD values at 450 nm were obtained using an ELISA plate reader  and the concentrations of cytokines were calculated according to the standard curve.+ cells were gated to analyze the expressions of CD40, CD80, CD86, and MHC II on DCs in all experiments.Annexin V/PI staining kit  was used to detect the viability of DCs upon CSE treatment and performed according to the manufacturer’s instruction. For analysis of DC maturation, cell surface staining was performed using the mAbs : PE-CD11c, APC-CD40, FITC-CD80, APC-CD11c, FITC-CD86, or PE-MHC II. All samples were collected on FACSCalibur (BD Biosciences) and the data were analyzed by the FlowJo platform . CD11cp < 0.05 was considered to be statistically significant.Statistical analysis was done by one-way analysis of variance (ANOVA).


Human immunodeficiency virus (HIV) capsid plays important roles at multiple stages of viral replication. At the initial stages, controlled uncoating (disassembly) of the capsid ensures efficient reverse transcription of the single-stranded RNA genome, into the double-stranded DNA. Whereas at later stages, a proper assembly of capsid ensures the formation of a mature infectious virus particle. Hence, the inhibition of capsid assembly and/or disassembly has been recognized as a potential therapeutic strategy, and several capsid inhibitors have been reported. Of these, PF-3450074 (PF74) has been extensively studied. Recently reported GS-CA inhibitors (GS-CA1 and GS-6207), have shown a strong potential and appear to contain a PF74 scaffold. The location of resistance mutations and the results of structural studies further suggest that GS-CA compounds and PF74 share the same binding pocket, which is located between capsid monomers. Additionally, phenylalanine derivatives containing the PF74 scaffold show slightly enhanced capsid inhibiting activity. A comparison of capsid structures in complex with host factors and PF74, reveals the presence of common chemical entities at topologically equivalent positions. Here we present the status of capsid inhibitors that contain PF74 scaffolds and propose that the PF74 scaffold may be used to develop strong and safe capsid inhibitors. Currently recommended antiretroviral therapies (cART) effectively suppress HIV and maintain HIV viral load below the detection levels (<50 copies/mL). As a result, HIV/AIDS is now a chronic and manageable disease ,3,4,5,6.The capsid monomer (CA) is a 24 kDa α-helical protein with two distinct domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a five residue flexible linker . During At the early stages of HIV-1 infection, i.e., after infection and the fusion of the viral envelope with the host cell membrane, capsid is released into the cytosol where it undergoes controlled disassembly (uncoating). While the timing, mechanism, and extent of capsid uncoating is controversial ,16,17,18Several capsid-binding small molecules, which inhibit viral replication have been identified ,50,51,5250s of 240 pM and 140 pM, respectively) [50s of 100 pM and 50 pM, respectively [Recently reported two capsid inhibitors, GS-CA1 b and GS-ctively) . Alternaectively . Both coectively ,62,63. AThe structure of GS-CA1/CA complex has been solved, but it is not available in the public domains such as the Protein Data Bank . HoweverIn vitro resistance selection experiments have identified mutations L56I, M66I, Q67H, N74D and A105E under GS-CA1 pressure . SeveralTogether with common structural moieties in GS-CA1, GS-6207 and PF74, the GS-CA compounds have additional chemical groups that have extensive interactions with the NTD of adjacent capsid monomer. More specifically, methanesulfonyl moiety of GS-CA1 and GS-6207 is within interacting distance of I37, P38, S41, I135 and N139 . It is pIn order to dissect the small molecule binding site between two capsid monomers of an hexamer, we employed the SiteMap program  of Schrö50/EC50) of 13.33 compared to the SI of PF74 (SI = 11.85) [The proposal that interactions of compounds with both capsid monomers involved in binding site formation may enhance potency of PF74-based molecules, was recently studied by synthesizing phenylalanine derivatives and testing their inhibitory activity . One of = 11.85) . These rAnother reported capsid inhibitor BI-2 that contains a partial PF-74 polyphenyl core binds at the PF74 binding site . A superCPSF6 and NUP153 are host factors that have been implicated in nuclear entry of HIV-1 through their interactions with capsid ,77,78,79The crystal structures of capsid bound to the peptides from CPSF6 (313–327) and NUP153 (1407–1423) have been reported . StructuOther reported capsid inhibitors include benzodiazepine (BD) and benzimidazole (BM) compounds ,83,84, CIn conclusion, here we present the current predicted mechanisms of capsid inhibitors containing PF74 scaffold. PF74, although used extensively in experimental settings, has poor metabolic properties suggesting that improved capsid inhibitors can be developed using PF74 as the starting point.