These responses are suppressed by dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). Here, we tested the hypothesis that dietary DHA supplementation suppresses cSiO2-induced inflammatory proteins in bronchoalveolar alveolar lavage fluid (BALF) and plasma of lupus-prone mice. Archived tissue fluid samples were used from a prior investigation in which 6 wk-old lupus-prone female NZBWF1 mice were fed isocaloric diets containing 0 or 10 g/kg DHA for 2 wks and then intranasally instilled with 1 mg cSiO2 or vehicle once weekly for 4 wks. Cohorts were terminated at 1, 5, 9 or 13 wk post-instillation (PI). BALF and plasma from each cohort were analyzed by high density multiplex array profiling of 200 inflammatory proteins. cSiO2 time-dependently induced increases in the BALF protein signatures that were highly reflective of unresolved lung inflammation, although responses in the plasma were much less robust. Induced proteins in BALF included chemokines , enzymes , adhesion molecules , co-stimulatory molecules , TNF superfamily proteins , growth factors , and signal transduction proteins , many of which were blocked or delayed by DHA supplementation. The BALF inflammatory proteome correlated positively with prior measurements of gene expression, pulmonary ectopic lymphoid tissue neogenesis, and induction of autoantibodies in the lungs of the control and treatment groups. Ingenuity Pathway Analysis (IPA) revealed that IL-1β, TNF-α, and IL-6 were among the top upstream regulators of the cSiO2-induced protein response. Furthermore, DHA’s effects were associated with downregulation of cSiO2-induced pathways involving i) inhibition of ARE‐mediated mRNA decay, ii) bacterial and viral pattern recognition receptor activation, or iii) TREM1, STAT3, NF-κB, and VEGF signaling and with upregulation of PPAR, LXR/RXR and PPARα/RXRα signaling. Altogether, these preclinical findings further support the contention that dietary DHA supplementation could be applicable as an intervention against inflammation-driven autoimmune triggering by cSiO2 or potentially other environmental agents.Repeated short-term intranasal instillation of lupus-prone mice with crystalline silica (cSiO Systemic lupus erythematosus (lupus), a chronic autoimmune disease predominantly affecting young women of child-bearing age, is caused by the loss of immunological tolerance resulting from yet poorly understood interactions between an individual’s genome and the environment , 2. Init2) exposure in construction, mining, and ceramics industries has been epidemiologically associated with lupus and other autoimmune diseases  can ameliorate chronic inflammatory and autoimmune diseases  and exte2-triggered autoimmune disease onset and progression in autoimmune-prone female NZBWF1 mice fed control diets or diets containing DHA at the realistic human caloric equivalent of 2 or 5 g/d. After 2 wk on these dietary regimens, mice were treated weekly for 4 wk with intranasally instilled cSiO2 (1 mg) and then sacrificed 1, 5, 9, and 13 wk. DHA supplementation dose-dependently reduced cSiO2-triggered B- and T-cell, follicular dendritic cell, and IgG+ plasma cell accumulation in the lungs along with development of glomerulonephritis. Subsequently, tissues from the aforementioned study were analyzed for 800 immune-related genes using targeted multiplex platform  responses, and antigen processing. Remarkably, DHA supplementation dose-dependently attenuated these responses, suggesting that consumption of this fatty acid impedes gene responses that play a central role in inflammation, ectopic lymphoid tissue neogenesis, and autoimmunity.To ascertain how ω-3 PUFAs influence environment-triggered lupus flaring, Bates and coworkers  comparedplatform . Intrigu2-induced gene expression by DHA influences the inflammatory proteome. We therefore conducted a targeted proteomic analysis of archived tissue fluids from the Bates et al. study  in 25 μl PBS or 25μl PBS vehicle (VEH) alone. Afterwards, mice were fed assigned diets for the experiment duration. Cohorts (n=8/group) were terminated at 1, 5, 9, and 13 wk post-instillation (PI) of the final cSiO2 dose and bronchoalveolar lavage fluid (BALF), blood, and organs were collected, processed, and stored at -800C as described previously  of BALF from 1, 5, 9, and 13 wk PI and plasma from 5, 9, and 13 wk PI were analyzed by RayBiotech  for the expression of 200 immune-related proteins using the Quantibody Array Q4000 . Inflammation-associated proteins on the array were identified and functionally classified by RayBiotech as: i) chemokines, ii) adhesion molecules, iii) co-stimulatory molecules, iv) enzymes, v) signal transduction proteins, vi) TNF superfamily, vii) growth factors, and viii) cytokines  and Principal Component Analysis (PCA) were visualized using ClustVis . For heat-tests for all proteins to determine difference in expression for cSiO2-exposed mice provided CON or DHA diet to the vehicle control (VEH/CON). Following this exploratory analysis, select proteins were further examined by scatter plot . The robust regression and outlier removal (ROUT) method was employed to discern outliers, which were excluded from further analysis (Q = 0.5%). Data were analyzed by two-way ANOVA for experimental factors time point and treatment and their interaction, with post-hoc Tukey HSD multiple comparisons test to determine the effect of treatment at each time point. If individual groups failed to pass at least one normality for Gaussian distributions tests , data were log-transformed.Values were centered by rows with imputation used for missing value estimation and rows were clustered using Euclidean distance and Ward linkage. Initial statistical analyses included pairwise Student’s cor and corrplot functions in R (www.R-project.org) or using Prism (GraphPad). A significant correlation was inferred when ρ > 0.5 or <-0.5 and p<0.05. To corroborate protein expression data, Spearman rank correlations were performed between protein expression data from this study and gene expression data previously reported for corresponding animals (2 fold-change in expression levels relative to the VEH/CON group. Log2 fold-changes in protein expression (expressed as MFI) relative to the VEH/CON group were calculated and correlated to the corresponding gene expression values. Spearman rank correlations for gene-protein interactions were determined using Microsoft Excel and bar graphs generated using GraphPad Prism.Spearman rank correlation analyses were performed using  animals , with bo+ (B cells), CD3+ (T cells), CD21/35+ (follicular dendritic cells), and lymphoid aggregates, expressed as percent of lung area analyzed, which are collectively indicative of ectopic lymphoid neogenesis  for each protein were submitted for IPA core analysis with the protein’s UniProt identifiers. Protein expression was compared against its corresponding gene/protein expression data in the Ingenuity Pathway Knowledge Base. Canonical pathways and upstream regulator analysis tools in IPA core analysis were used at default setting to explore cSiO2 triggered immune-related pathways and predict expression of upstream regulators. The results from canonical pathways and upstream regulators analysis for different treatments groups were compared using comparison tool in IPA and visualized with GraphPad Prism. Proteins in data set with Z-scores ≥ 2 or ≤−2 with an overlapping p-value <0.05 were considered to be significantly activated or significantly inhibited, respectively, for all analyses or predictions.Protein interactions and functional networks in the data set were determined using Ingenuity Pathway Analysis . Calculated log2 in CON-fed lupus-prone NZBWF1 induced increased expression of a diverse group of proteins in BALF relative to baseline over time that were highly reflective of unresolved inflammation in the lung, with significant elevations in 65, 60, 82, and 82 proteins being observed at 1, 5, 9, and 13 wk PI, respectively , enzymes , adhesion molecules , co-stimulatory molecules , TNF superfamily proteins , growth factors , and signal transduction proteins  in the BALF  or 9 wk PI (CD6)  or later at 9 wk PI   with semiquantitative histopathology severity scores of minimal (1 out 4) and mild to moderate (2/3 out of 4) at 1/5 and 9/13 wk PI, respectively  had minimal alveolitis at 1,5, and 9 wk PI and only mild alveolitis at 13 wk PI. Total inflammatory cell numbers in BALF of these DHA-supplemented mice were similarly suppressed at 9 and 13 wk PI  are particularly pertinent in ELT neogenesis. BLC, which is produced by follicular dendritic cells, T follicular helper cells, and Th17 cells, is essential for B-cell localization into lymphoid follicles. Other CXCL chemokines might also impact cSiO2-induced autoimmunity. MIP-2 (CXCL3) primarily recruits neutrophils to sites of inflammation that, upon degranulation, could secrete proteolytic enzymes that elicit bystander tissue injury and inflammation, or release neutrophil extracellular DNA traps via NETosis, a source of self-DNA and autoantigens , a family of enzymes that degrade extracellular matrix and basement membrane components, is consistent with prior in vivo and in vitro investigations -selectin (CD62L) is expressed on neutrophils, monocytes, and most lymphocytes .LR: data curation, data analysis/interpretation, figure preparation, manuscript writing. MB: study design, animal study, necropsy, immunohistochemical analyses and morphometry, manuscript preparation and manuscript editing. AB and KW: data analysis/interpretation, figure preparation, manuscript editing. JH: study design, lung/kidney histopathology, morphometry, data analyses. JP: study design, coordination, oversight, funding acquisition, manuscript preparation and submission. All authors contributed to the article and approved the submitted version.This research was funded by NIH ES027353 (JP), NIH F31ES030593 (KW), NIH T32ES007255 (KW), Lupus Foundation of America , USDA National Institute of Food and Agriculture Hatch Projects 1020129 (JP) and UTA-01407 and UTA-01456 (AB), and the Dr.Robert and Carol Deibel Family Endowment (JP).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Corchorus olitorius L. cv. O-9897) by exposing twenty-day-old seedlings to three doses of salt . Biochar was pre-mixed with the soil at 2.0 g kg−1 soil, and chitosan-100 was applied through irrigation at 100 mg L−1. Exposure to salt stress notably increased lipid peroxidation, hydrogen peroxide content, superoxide radical levels, electrolyte leakage, lipoxygenase activity, and methylglyoxal content, indicating oxidative damage in the jute plants. Consequently, the salt-stressed plants showed reduced growth, biomass accumulation, and disrupted water balance. A profound increase in proline content was observed in response to salt stress. Biochar and chitosan supplementation significantly mitigated the deleterious effects of salt stress in jute by stimulating both non-enzymatic  and enzymatic  antioxidant systems and enhancing glyoxalase enzyme activities  to ameliorate reactive oxygen species damage and methylglyoxal toxicity, respectively. Biochar and chitosan supplementation increased oxidative stress tolerance and improved the growth and physiology of salt-affected jute plants, while also significantly reducing Na+ accumulation and ionic toxicity and decreasing the Na+/K+ ratio. These findings support a protective role of biochar and chitosan against salt-induced damage in jute plants.We investigated the role of biochar and chitosan in mitigating salt stress in jute ( Plants face several types of environmental stresses, such as salinity, drought, waterlogging, metal/metalloid toxicity, extreme temperature, and mechanical injury during their growth period. These stresses have unfavorable effects on crop productivity and therefore threaten global food security . Among m+ and Cl− ions in the root zone, and this creates an imbalance in the osmotic potential of cells and reduces water uptake capacity. High amount of Na+ ions are also accumulated in plants under saline conditions, and this inhibits K+ uptake, thereby retarding several K+-dependent physiological processes, such as membrane equilibrium, turgor potential, enzyme activities, photosynthesis, transpiration, and protein synthesis [2•−; hydrogen peroxide, H2O2; and singlet oxygen, 1O2), which disrupts the equilibrium of antioxidant defense and overwhelms their capacity to detoxify ROS. Ultimately, the excess generation of ROS causes oxidative damage and death of the plant [Plants under salt stress are exposed to increased concentrations of Naynthesis ,6,7. Salhe plant . Plants he plant .S-transferase (GST), and glutathione peroxidase (GPX), which aid in scavenging the ROS [The plant antioxidant defense system consist of non-enzymatic antioxidants, such as ascorbate (AsA), glutathione (GSH), tocopherols, flavonoids, and carotenoids, and enzymatic antioxidants, such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione  the ROS . Methylg the ROS . Plants  the ROS ,10. Rece the ROS ,12.+ ions on its exchange sites, while also increasing the soil K+ ion availability to maintain a consistent Na+/K+ ionic balance and help to reduce Na+ uptake in plants [Biochar (BC) is a pyrolyzed product of various agricultural farm residues  and contains profound amount of organic carbon, along with calcium, magnesium, and organic carbonates ,14. Biocn plants .Chitosan (CHT) is a linear polysaccharide produced through deacetylation of the chitin component of the exoskeleton of several crustaceans . CHT is Corchorus olitorius L.), a crop plant that is widely cultivated on the Asian and African continents for its bast fibers, which are used in a wide range of marketable products [One plant that is adversely affected by salt stress is jute  were collected from Bangladesh Jute Research Institute and sterilized. Sun-dried soil was mixed with organic manure, urea, muriate of potash, triple superphosphate, zinc sulfate, and gypsum, according to Islam [−1 soil 7 days before seed sowing in each pot. At 20 days after sowing (DAS), one set of plants was irrigated with various doses of salt  and always maintained field capacity by irrigating with 500 mL of water or salt solution at each 5-day interval. For CHT application, a concentrated solution of chitosan-100  was prepared by dissolving in 1% acetic acid and pH of the solution was neutral. Then, at 20 and 30 DAS during irrigation, the plant with either water or salt solution, CHT was applied at 100 mg L−1 solution, but not applied in the BC-amended soil. At 40 DAS, several morphological, physiological, and biochemical parameters were measured. The treatments were replicated three times.Mature, healthy seeds of jute .2O) to remove the adhering soil with roots. After that, roots were separated from shoots, and the fresh weight (FW) of roots and shoots was measured by a digital balance, then oven-dried at 70 °C for 72 h and weighed again for dry weight (DW). Finally, the weights were averaged and expressed as g plant−1.Five randomly selected plants from each treatment were uprooted and washed with distilled water  reading was taken to measure the total chlorophyll (Chl) content by using a SPAD meter . Values of each treatment were averaged for SPAD value.2O in Petri dish for 24 h by covering it with filter paper and kept in dark. After removing the adhering excess water, the leaf lamina was weighted again for the turgid weight (TW). These leaf laminas were dried in an electric oven for 48 h by maintaining 70 °C and weighted for DW. Finally, RWC was estimated following the equation:Using the formula of Barrs and Weatherly , leaf reg for 12 min. Then the supernatant was heated in a water bath for 60 min at 100 °C after mixing with acid ninhydrin and glacial acetic acid. After that, the mixture was cooled down at room temperature and toluene was added and vortexed, and then the upper chromophore was subjected to spectrophotometer at 520 nm. The absorbance reading was finally plotted in a standard curve for Pro determination.Following the method of Bates et al. , 0.5 g fg, 15 min). Then the supernatant was mixed with thiobarbituric (TBA) acid reagent containing 0.5% TBA and 20% TCA and heated for 30 min at 95 °C. After cooling, absorbance was recorded at 532 nm and non-specific absorbance was recorded at 600 nm, and finally, calculation was done by deducting the values of non-specific absorbance and an extinction coefficient of 155 mM−1 cm−1 was also used [For estimating malondialdehyde (MDA) content, 0.5 g leaf sample was homogenized into 5% trichloroacetic acid (TCA) and centrifuged  was homogenized in TCA and followed by centrifugation. Then the aliquot was incubated for 1 h after adding potassium-phosphate (K-P) buffer  and potassium iodide (1 mM). Then the absorbance was measured by using a spectrophotometer at 390 nm .2O. After that, the tubes were heated into a water bath at 40 °C for 1 h, and then the electrical conductivity (EC1) was measured by using electrical conductivity meter  cooling up to room temperature. Then again, the falcon tubes were heated at 121 °C for 40 min by using autoclave and after cooling, final electrical conductivity (EC2) was measured. Then, EL was calculated as follows:Leaf electrolyte leakage (EL) was measured by following the method of Dionisio-Sese and Tobita . Leaf sa2O2 and O2•−, 0.1% solution of 3,3′ diaminobenzidine (DAB) and nitro blue tetrazolium chloride (NBT) was used maintaining a pH of 3.8. After incubating for 72 h in dark, leaves were thoroughly cleaned with boiling 70% ethanol to remove the solution and then a picture was taken using a digital camera [For detecting Hl camera .2O instead of DTT used for AsA, and absorbance was taken at 265 nm. A standard curve was used to calculate the total AsA and AsA, and dehydroascorbate (DHA) content was estimated by subtracting the values of AsA from total AsA [An amount of 0.5 g leaf sample was homogenized in 5% metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) followed by centrifugation. Neutralization of aliquot was done by using 0.5 M K-P buffer (pH 7.0) and 0.1 M dithiothreitol (DTT) for determining total AsA and DHotal AsA .Following the method of Hasanuzzaman et al. , glutathw/v) glycerol following the method of Hasanuzzaman et al. [For enzyme extraction, 0.5 g leaf sample was homogenized in an ice-cooled mortar pestle by using extraction buffer containing 1 mM L-ascorbic acid (AsA), 100 mM KCl, 100 mM 0.5 M K-P buffer (pH 7.0), 5.0 mM β-mercaptoethanol, and 10%  activity was estimated by using an assay buffer containing 15 mM K-P buffer (pH 7.0), 0.5 mM AsA, 0.1 mM EDTA, HM−1 cm−1 .−1 cm−1) [For monodehydroascorbate reductase  activity, 50 mM Tris-HCl (pH 7.5), 2.5 mM AsA, 0.2 mM NADPH, ascorbate oxidized (AO), and enzyme was assayed at 340 nm in spectrophotometer, and calculation was done by using an extinction coefficient (6.2 mM−1 cm−1) .−1 cm−1.Dehydroascorbate reductase  was estimated according to Nakano and Asada  in which−1 cm−1 as extinction coefficient [Glutathione reductase  activity was assayed by a reaction buffer comprising K-P buffer (pH 7.0), EDTA, GSSG, NADPH, and enzyme. The absorbance was measured at 340 nm and the value was estimated by 6.2 mMfficient .Superoxide dismutase  activity was measured by following the method of El-Shabrawi et al. . Here, K2O2 (15 mM) and K-P buffer  following Hasanuzzaman et al. [−1 cm−1.Catalase  activity was measured by degrading the enzymes with Hn et al. . Absorba2O2, and the activity was observed at 470 nm. Extinction coefficient of 26.6 mM−1 cm−1 was used for calculation [Peroxidase  activity was measured by oxidation of guaiacol in presence of K-P buffer and Hculation .S-transferase  activity was assayed by a buffer comprising K-P buffer , GSH (1.5 mM), and 1-chloro-2,4-dinitrobenzene . The activity was observed at 340 nm and an extinction coefficient (9.6 mM−1 cm−1) was used for computing the activity [Glutathione activity .3, 1 mM), NADPH (0.12 mM), GSH (2 mM), GR (1 unit), and H2O2 (10.50 mM) with enzyme was spectrophotometrically observed at 340 nm. For computation of the activity, an extinction coefficient (6.62 mM−1 cm−1) was used.Glutathione peroxidase  activity was estimated by following the method of Elia et al. (2003) with slight modifications from Hasanuzzaman et al. . Assay b4; 16 mM), GSH (100 mM), and MG (35 mM) and observed at 240 nm. For calculating the activity, 3.37 mM−1 cm−1 was used as the extinction coefficient [Glyoxalase I  activity was assayed by a buffer which contained K-P buffer (pH 7.0), magnesium phosphate (MgSOfficient .S-D-lactoylglutathione (SLG), and enzymes were spectrophotometrically observed at 412 nm, and calculation was done by using 13.6 mM−1 cm−1 as an extinction coefficient.According to the procedure of Principato et al. , Glyoxalg. Then the extracted sample was again centrifuged with activated charcoal and neutralized by using saturated sodium carbonate. After that, neutralized sample was mixed with sodium-phosphate (Na-P) buffer and N-acetyl-L-cysteine and kept for 15 min to form N-α-acetyl-S-(1-hydroxy-2-oxoprop-1-yl) cysteine which was observed spectrophotometrically at 288 nm. Finally, MG content was calculated by using a standard curve.According to Wild et al. , plant s+ and K+ contents were measured with a Na+ meter  and K+ meter , respectively. A fresh leaf was washed with distilled water and then squeezed to extract the sap. The sap was applied directly to the sensor of the ion meter, and data values were expressed as ppm.Leaf Nap ≤ 0.05 was considered statistically significant.Statistical analyses were done using CoStat v.6.400 computer-based software  . The meaReduction of root length was noticed in the salt-stressed plant, in comparison to untreated control . Amendment with BC has improved root length in salt-treated plants, and the highest increase of root length was observed at 50 mM NaCl treated plants by 10% compared to the corresponding salt-stressed plants only. On the other hand, CHT supplementation increased root length by 8% in 150 mM NaCl stressed plant, in comparison to the salt-treated plants alone A.Shoot length was reduced by 41, 51, and 76% upon exposure to 50, 100, and 150 mM NaCl, respectively, in comparison to the untreated control . Under 50 mM NaCl-stressed plants, BC application improved the shoot length by 15%, whereas it was improved by 8% with CHT supplementation in comparison to the respective salt-stressed plant alone B.The stem diameter of salt-treated plant with or without BC and CHT supplementation showed a statistically similar result compared to untreated control  C.Exposure to 50, 100, and 150 mM NaCl reduced the root FW by 61, 119, and 201%, respectively, and reduced the shoot FW by 65, 95, and 166%, respectively, compared to the unstressed control plants. Supplementation with BC and CHT improved the root FW when compared to the corresponding salt-stressed but unsupplemented plants A,B.Exposure to 50, 100, and 150 mM NaCl stress decreased the root DW by 40, 64, and 104%, respectively, and the shoot DW by 47, 64, and 79%, respectively, compared to the unstressed control plants. Supplementation with BC improved the DW of the salt-stressed plants when compared to the corresponding salt-stressed but unsupplemented plants. CHT application enhanced the root DW by 18%, 16%, and 18% in plants treated with 50, 100, and 150 mM NaCl, respectively, when compared to the corresponding salt-stressed but unsupplemented plants. CHT amendment also improved the shoot DW (by 15%) in plants exposed to 75 mM NaCl compared to salt treatment alone C,D.Leaf RWC declined in salt-stressed plants compared to the control plants. Exposure to 50, 100, and 150 mM NaCl reduced the RWC by 18, 24, and 30%, respectively, in comparison to unstressed control plants. When compared to the corresponding salt-stressed but unsupplemented plants, supplementation with BC increased the RWC by 8%, 8%, and 10% in plants treated with 50, 100, and 150 mM NaCl, respectively, whereas CHT supplementation improved RWC by 6%, 7%, and 9%, respectively A.A sharp increase in Pro content was observed at the different levels of NaCl stress. Proline content notably increased by 67%, 77%, and 84% by 50, 100, and 150 mM NaCl, respectively, when compared to the untreated control plants. When compared to the corresponding salt-stressed but unsupplemented plants, BC supplementation reduced the Pro content by 33, 27, and 23% in plants treated with 50, 100, and 150 mM NaCl, respectively, while CHT supplementation decreased the Pro content by 21%, 14%, and 16%, respectively B.The greatest reduction in the SPAD value (by 10%) was observed in plants treated with 150 mM NaCl compared to the untreated control. When compared to the corresponding salt-stressed but unsupplemented plants, BC supplementation of plants treated with 100 mM and 150 mM NaCl increased the SPAD value by 10% in both cases, and supplementation with CHT increased the SPAD value by 10% in the plants treated with 100 mM salt C.2O2, LOX activity, and EL (%) in jute leaves. The MDA content increased by 24%, 36%, and 49% in plants exposed to 50, 100, and 150 mM NaCl, respectively, compared to the untreated control plants .The increases in the EL (%) in jute leaves due to salt stress indicated membrane damage. The EL increased by 25%, 34%, and 39%, respectively, following exposure of plants to 50, 100, and 150 mM, respectively, compared to the untreated control plants D. When c2O2 (brown spots) and O2•− (dark blue spots) in salt-stressed jute leaves, and the spots were reduced in size and number in the salt-stressed plants supplemented with BC and CHT , POD activity , GST activity , and GPX activity , whereas SOD activity increased  in plants exposed to 50, 100, and 150 mM NaCl stress, respectively, compared to the untreated control plants. The highest SOD activity was observed with BC supplementation (by 13% and 12%) and CHT supplementation (by 13% and 12%) in plants treated with 50 and 100 mM NaCl, respectively, compared to the corresponding salt-stressed but unsupplemented plants. The SOD activity was statistically similar following BC or CHT supplementation in plants exposed to 150 mM NaCl. BC supplementation improved the CAT activity by 17%, 19%, and 32% in plants exposed to 50, 100, and 150 mM NaCl, respectively, and CHT significantly increased the CAT activity by 13% in plants exposed to 50 mM salt. BC supplementation also enhanced POD activity by 19%, 20%, and 24% in plants exposed to 50, 100, and 150 mM salt, respectively, compared to the corresponding salt-stressed but unsupplemented plants. CHT supplementation improved the POD activity by 16% and 20% in plants exposed to 50 and 100 mM salt stress compared to the corresponding salt-stressed but unsupplemented plants. BC supplementation increased the activity of GST by 14%, 21%, and 25% and GPX by 16%, 22%, and 26% in plants exposed to 50, 100, and 150 mM NaCl, respectively, compared to the corresponding unsupplemented salt-stressed plants. CHT supplementation increased GST activity by 14%, 16%, and 20% and GPX activity by 13%, 15%, and 14% in plants treated with 50, 100, and 150 mM NaCl, respectively, compared to the corresponding salt-stressed but unsupplemented plants A–E.The MG content notably increased, whereas the activity of Gly I and Gly II decreased in salt-stressed plants compared to the untreated control plants. BC and CHT supplementation decreased the MG content in salt-stressed plants. BC supplementation increased the activity of Gly I  and Gly II  in plants exposed to 50, 100, and 150 mM salt, respectively, while CHT supplementation enhanced the activity of Gly I by 19%, 22%, and 15% and Gly II by 16%, 14%, and 13%, respectively, compared to the corresponding salt-stressed but unsupplemented plants A–C.+ content and reduced the K+ content in the leaves of jute plants. Pronounced accumulation of Na+ content (by 81%) and reduction of K+ content (by 72%) were observed in plants treated with 150 mM NaCl compared to the untreated control plants. Similarly, the Na+ content increased by 51% and 75% in plants treated with 50 mM and 100 mM salt, while the K+ content decreased by 50% and 72%, respectively, compared to the untreated control plants. However, the reduction in Na+ content and the increase in K+ content resulted in an increase in the Na+/K+ ratio in the BC- and CHT-supplemented plants compared to the salt-stressed but unsupplemented plants  and LOX activity (associated with lipid disintegration), and all these responses increase the EL under salt stress. The present findings agree with the previous studies by Hasanuzzaman et al. [2O2, LOX activity, and EL with BC amendment have been reported in maize varieties by Alharby et al. [2O2 and MDA contents in response to CHT treatment [2O2, and EL declined with the application of CHT in salt-stressed sweet peppers. Amendment with BC, CHT, or their combination decreased the MDA and H2O2 contents and protected lentil plants from Ni-induced oxidative damage [Salt stress exerts its negative effects on plant physiology and metabolic processes by inducing osmotic stress, ionic toxicity, and water and nutrient imbalances and by accelerating ROS accumulation, which collectively threatens the antioxidant defense mechanisms of plants . A certan et al. , Siddiqun et al. , Rahman n et al. , and Parn et al. . Reductiy et al. . Similarreatment . AlKahtareatment  reportede damage .The non-enzymatic antioxidants AsA and GSH play pivotal roles in scavenging ROS through the AsA-GSH cycle, which helps to protect cellular organelles and biomolecules from oxidative damage. AsA is a strong antioxidant and electron donor, so it neutralizes ROS by delocalization of electrons, and it also participates in the biosynthetic pathway of some phytohormones. In contrast, GSH is a non-protein thiol compound that scavenges ROS through the AsA-GSH cycle and maintains redox homeostasis . Salt st2O2) and for regenerating AsA and GSH. In the AsA-GSH pool, APX participates in scavenging H2O2 by producing monodehydroascorbate (MDHA), which is transformed into AsA in the presence of MDHAR. In another non-enzymatic reaction, MDHAR is reduced into DHA to regenerate AsA in the presence of DHAR (a GSH-dependent enzyme). The reduced form of GSH, which was previously oxidized to GSSG, is then regenerated through the action of GR [Four key enzymes—APX, MDHAR, DHAR, and GR—are directly involved in the cycle that links the AsA-GSH cycle for scavenging ROS . Subsequent conversion of H2O2 to H2O and O2 depends on the enzyme CAT [2O2 and other hydroperoxides. Other xenobiotics and electrophilic substances are also degraded into less toxic compounds by the action of the GST and GPX enzymes and are ultimately sequestered in vacuoles [2O2 by enhancing CAT and POD activity with CHT amendment resulted in increased tolerance of milk thistle plants to salinity [The frontline of defense in scavenging ROS in plants is the SOD enzyme, a metalloenzyme that catalyzes a dismutation of Ozyme CAT ,64. In tzyme CAT ,57. Glutvacuoles . The actvacuoles . Sofy etvacuoles  also repsalinity . Foliar salinity . Likewissalinity . Notablesalinity . A profosalinity . Similarsalinity . The actsalinity .Methylglyoxal is a cytotoxic compound that increases greatly in level in response to environmental stresses. Plants are protected from MG-induced cellular damage by the action of glyoxalase enzymes (Gly I and Gly II), which play a crucial role in the detoxification of MG by a two-step reaction. MG is first converted into SLG via GSH in the presence of the Gly I enzyme, and then D-lactic acid is produced from SLG through the enzymatic action of Gly II . In the + and Cl− concentrations across the root zone and disrupts the ionic homeostasis in plants, while also reducing water and nutrient uptake capacity from the soil solution. The presence of increased amounts of soluble ions in the soil medium increases the transportation of Na+ ions to various parts of the plants. Accumulation of Na+ ions decreased the K+ content in the jute plants in the current study, and K is considered an essential nutrient for carrying out different metabolic activities. The results presented here agree with other findings in plants under salt stress [+ and Cl−) and increased K+ uptake, thereby helping to maintain ion homeostasis under salt stress. Similarly, CHT can also bind Na+ and Cl− ions and improve K+ mobility in plants. Yang et al. [+ content and increased K+ availability in quinoa under saline conditions. Similarly, a reduced Na+ accumulation has been observed following the application of BC and CHT under salt stress conditions in soybean [+ and augmented K+ uptake, thereby reducing ion toxicity by balancing the Na+/K+ ratio in salt-stressed wheat plants.Salt stress increases Nat stress ,45,57. Sg et al.  demonstr soybean . Hafez e soybean  mentione2O2 and MDA content), EL, and LOX activity were increased with increasing NaCl concentrations. Amendment with BC and CHT increased the RWC and reduced the Pro content, while also reducing the oxidative damage by decreasing the MDA and H2O2 content and the EL and LOX activity in salt-stressed jute plants. Salt stress decreased the AsA and GSH content, but increased the DHA and GSSG content, thereby reducing the AsA/DHA and GSH/GSSG ratios. However, BC and CHT supplementation enhanced the accumulation of AsA and GSH, followed by decreases in the content of DHA and GSSG, resulting in increased AsA/DHA and GSH/GSSG ratios. Salt stress decreased the MDHAR, DHAR, GR, CAT, POD, GST, and GPX activities in jute plants, but increased the APX (up to 100 mM NaCl stress) and SOD activities. Amendment with BC and CHT improved the defense responses of jute plants by enhancing antioxidant enzyme activity. MG accumulation increased and Gly I and Gly II activity decreased in response to salt stress, whereas BC and CHT supplementation enhanced Gly I and Gly II activity to support MG detoxification. The Na+ ion content increased under salt stress and reduced K+ accumulation. However, BC and CHT amendment decreased the Na+ accumulation and enabled K+ uptake under salt stress. Taken together, the results obtained in the current study confirm that both BC and CHT are capable of mitigating salt-induced oxidative stress by modulating the ROS metabolism, antioxidant defense system, and glyoxalase system to improve plant growth and physiology under salt stress (In summary, exposure of jute plants to salt stress decreased plant growth, biomass accumulation, stem diameter, and photosynthetic pigment contents, but these decreases were mitigated by the exogenous supplementation of BC and CHT. The RWC decreased, whereas the Pro content increased, to protect the plants from the osmotic stress created by saline conditions. Oxidative stress indicators (Ht stress .The present investigation evaluated the role of BC and CHT as protectants from salt-induced damage in jute plants. Under salt stress, the jute plants showed responses indicative of ionic and osmotic stress, ROS over-accumulation, lipid peroxidation, enhanced LOX activity, MG accumulation, growth reduction, and impaired physiobiological activities. Supplementation with BC and CHT enhanced the activities of both non-enzymatic and enzymatic antioxidants to mitigate ROS-induced damage. Supplementation with BC and CHT decreased MG toxicity by upregulating Gly I and Gly II activity while also maintaining ion homeostasis and osmotic balance. Overall, the SPAD value, RWC, growth parameters, and phenotype appearance were improved by BC and CHT supplementation under salt stress. However, further investigations should be carried out to explore the actual mechanisms by which BC and CHT perform protective roles and to elucidate the complex interaction between the different biochemical pathways involved in salt stress tolerance.


The tumor immune microenvironment (TIME) has largely been reported to cooperate on tumor onset and progression, as a consequence of the phenotype/functional plasticity and adaptation capabilities of tumor-infiltrating and tumor-associated immune cells. Immune cells within the tumor micro  and macro  environment closely interact by cell-to-cell contact and/or via soluble factors, also generating a tumor-permissive soil. These dangerous liaisons have been investigated for pillars of tumor immunology, such as tumor associated macrophages and T cell subsets. Here, we reviewed and discussed the contribution of selected innate immunity effector cells, namely neutrophils and natural killer cells, as “soloists” or by their “dangerous liaisons”, in favoring tumor progression by dissecting the cellular and molecular mechanisms involved. Neutrophils are the most abundant circulating leukocytes, accounting for 50–70% of blood cells. During neutrophil maturation from common myeloid progenitors, the granulocyte-monocyte progenitor cells are the first to acquire the neutrophil lineage marker CD66b . Then, dNeutrophils represent the first-line defense against infections, thus they are rapidly recruited from the bloodstream to the site of injury where they eliminate pathogens, particularly bacteria, by phagocytosis, degranulation, and release of extracellular traps (NETs) ,5,6,7.Although the functional diversity of neutrophils has been largely overlooked, compared with other myeloid cells, several studies have recently pointed out a high heterogeneity among them, also highlighting their plasticity . It has In cancer, mimicking the dichotomy between pro-inflammatory/M1-like and anti-inflammatory/tissue-repairing/TAM/M2-like macrophages, neutrophils have been conventionally termed N1 and N2 neutrophils, with the latter characterized by immunosuppressive, pro-metastatic, and pro-angiogenic activities ,14. Several studies support the pro-tumoral properties of N2 neutrophils, that predominantly express CC and CXC chemokines such as CCL2, 3, 4, 8, 12, and 17 and CXCL1, 2, 8, and 16, in addition to higher expression of vascular endothelial growth factor (VEGF), CXCR4 and matrix metalloproteinase 9 (MMP9) ,15,16 FB. MolecuMoreover, several studies  have shohigh, CCL3high, ICAM1high, and arginaselow expression [Regarding the anti-tumor potential of N1 cells, it has been shown that “cytotoxic neutrophils” are characterized by TNFαpression ,20  cells are large granular lymphocytes from innate immunity, participating in the early recognition and eliminations of virus-infected and malignant-transformed cells. Currently, NK cells are classified as innate lymphoid cells (ILCs) and originate from common innate lymphoid progenitors in the bone marrow. Subsequently, they migrate to different lymphoid tissues or non-lymphoid tissues, where they are “educated”, acquiring phenotype and functions typical of tissue residency. The NK inhibitory receptor repertoire is adapted to the MHC class I molecules borne by the host, assuring NK cell tolerance against self-cells. On the other hand, NK cells are simultaneously stimulated by activating receptors that trigger NK cell responses. In the presence of healthy cells, activating signals are low, thus the binding of inhibitory receptors to MHC class I molecules is sufficient to induce NK cell tolerance. In contrast, when NK cells recognize altered cells  that lack or downregulate MHC I expression, the activating signals overcome the inhibitory ones, leading to the killing of altered cells . dimCD16+ (more mature phenotype) NK cells account for 90–95% of circulating NKs and exert their killing function by releasing perforin, granzymes, and mediating ADCC (brightCD16− NKs (considered less mature NKs) account for 5–10% of circulating NKs and act both as distant killers and regulatory cells, by producing TNFα and IFNγ  and CD16  process) surface antigens, two major NK cell subsets have been characterized. CD56ing ADCC C. CD56brand IFNγ D, which superbright CD16− cells and support the formation of the spiral arteries, by producing VEGF, placental growth factor (PlGF), and CXCL8 (IL8) , which are identified as CD56L8 (IL8) D. dNK ceEven if little NK-cell infiltration has been reported in solid tumors, NK cell infiltration has been observed in several types of cancers, including melanoma , gastro-bright with impaired cytotoxic functions [brightCD9+CD49a+ phenotype [Several studies demonstrated a relevant role for NK cells in cancer progression and patient outcome ,34. Despunctions ,40,41. Sunctions ,43,44. Ihenotype ,46,47. Ahenotype ,46,47 anhenotype . Mechanihenotype  and the In line, Gotthardt et al., highlighted the relationship between NK cells and angiogenesis, demonstrating that Stat5∆/∆Ncr1-iCreTg-Vav-Bcl2 mice failed to control tumor growth of RMA-S lymphoma cells and a v-abl transformed tumor, clarifying the significant tumor-promoting function of STAT5-deficient NK . FurtherThis evidence suggests that bi-directional interplay between NK cells and neutrophils act as a relevant hub in shaping the TME and the possible response to specific (immune)therapies, finally placing this dangerous liaison as a new potential immune-related hallmark of cancer.Several studies have demonstrated that TANs can directly or indirectly  contribute to the generation of an immunosuppressive TME. Studies of neutrophil-induced immunosuppression have mostly focused on their ability to inhibit T cell functions ,51. HereIn a colorectal cancer murine model, generated by CT-26 cells intramuscularly injected into the flanks of BALB/c mice, neutrophils have been shown to suppress the NK cell infiltration, by downregulating CCR1 and to impair anti-tumor capabilities A by cellNeutrophils isolated from the CT-26 tumor-bearing mice, when co-cultured both with naïve and tumor-bearing NK cells, displayed a decreased production of IFNγ; treatment with PD-L1 neutralizing antibody was effective in limiting tumor-bearing neutrophil inhibitory effects on tumor-bearing NK cells, but did not exert the same inhibitory effect on naïve NK cells . AdditioThe NKp46 NCR is considered a key molecule for NK-related killing capacity; Valayer et al., showed that NKp46 decreasing on NK cells is related to activated neutrophil-derived serine proteases and in particular, cathepsin G (CG) is responsible for NKp46 extracellular cleavage  Figure Ci, thus dim NK cells [bright NK cells, in which NKp46 was increased, probably due to high anti-oxidative intrinsic capability [Neutrophils can activate or suppress the NK cells cytotoxic functions and therefore can indirectly interfere with the angiogenic process. Romero et al., demonstrated that neutrophil-derived ROS can modulate NKp46 expression on NK targeted cells  Figure Cii. IndeNK cells   injected with 4T1 mammary carcinoma cells, showed that neutrophils can suppress intraluminal NK-mediated tumor cell elimination and enhance extravasation of disseminated carcinoma cells .scid mice were more prone to develop lung metastases, whereas NOD-nullscid IL2rγ mice, which also lack NK cells, are characterized by a reduced number of metastases [Using E0771 tumor bearing mice, Li et al., reported that neutrophils either promote or inhibit metastasis, depending on the presence of NK cells . They shtastases . Converstastases .Neutrophils represent one of the first infiltrating cell type within TME and can shape TME promoting tumor growth and metastasis formation by angiogenesis stimulation ,63. In aAngiogenesis stimulation can be mainly mediated by the release of soluble factors including the main pro-angiogenic master regulator VEGF ,66. VessNK cells can play a dual role related to angiogenesis by acting as the inhibitor and promoter of this process. In response to IL12 , cytotoxIndeed, IL12 increased the production of cytolytic mediators as granzyme and perforin by NK cells and blocked the pro-angiogenic effect of FGF2 by IP-10 (CXCL10) A, causinThe anti-angiogenic loop that involves IP-10 can be driven by both NK cells and neutrophils that together can contribute to angiogenesis inhibition. The anti-angiogenic effect of IFNγ can also be boosted by other neutrophil-derived cytokines as IL12, IL15, and IL18 ,76 FiguA that, dCXCL8 can work as a bridge-molecule between NK cells and neutrophils. CXCL8 can target neutrophils, increasing their recruitment, which in turn, sustains angiogenesis together with tumor progression in Ras oncogene driven tumors . In addi+ neutrophils are responsible for angiogenesis stimulation since expressed high levels of genes encoding for VEGF and MMP9, together with transcription factor c-myc and STAT3, which are positive regulators of both VEGF and MMP9 [Activated NK cells increased CD11b expression on neutrophils and promoted CD62L (L-selectin) shedding through IFNγ and granulocyte-macrophage colony stimulating factor (GM-CSF) release  Figure B. In a tand MMP9   but also the dangerous liaisons occurring between different immune cells in the tumor micro- and macro-environments, represent crucial events impacting cancer progression and the success of the therapeutic regimens. Based on these concepts, immunotherapy has emerged as the new next-generation approach in cancer therapy, with different success in some cancer types  and with minor or null success in other cancer types. The still persistent window of “non-successful” immunotherapy further opens the need to deepen the investigation into the immune cell–TME interactions in cancer. This is a crucial point in perspective/future immunotherapeutic approaches, where modifying/re-educating the immune system should take into consideration not only the action on altered immune cells such as soloist elements, but also on the immune cells/TME interactions.


KRAS (RASMUT) reduces therapeutic options, negatively affecting prognosis of the patients. In this setting, administration of CDK4/6-inhibitors, alone or in combination with other drugs, is being tested as promising therapeutic strategy. Identifying sensitive patients and overcoming intrinsic and acquired resistance to CDK4/6 inhibition represent still open challenges, to obtain better clinical responses. Here, we investigated the role of the CDK inhibitor p27kip1 in the response to the selective CDK4/6-inhibitor Palbociclib, in colorectal cancer. Our results show that p27kip1 expression inversely correlated with Palbociclib response, both in vitro and in vivo. Generating a model of Palbociclib-resistant RASMUT colorectal cancer cells, we observed an increased expression of p27kip1, cyclin D, CDK4 and CDK6, coupled with an increased association between p27kip1 and CDK4. Furthermore, Palbociclib-resistant cells showed increased Src-mediated phosphorylation of p27kip1 on tyrosine residues and low doses of Src inhibitors re-sensitized resistant cells to Palbociclib. Since p27kip1 showed variable expression in RASMUT colorectal cancer samples, our study supports the possibility that p27kip1 could serve as biomarker to stratify patients who might benefit from CDK4/6 inhibition, alone or in combination with Src inhibitors.In colorectal cancer, mutation of  RAS mutations (RASMUT), which determines a constitutive activation of Ras/Raf/MEK/ERK signaling pathway. RASMUT CRC are intrinsically resistant to anti-epidermal growth factor receptor  represents the third most common cancer and the second leading cause of cancer-related death worldwide , 2. Abou therapy , 2. Simi success –7. Incre success , 9 that pression , 11.MUT CRC patients, who currently have very few therapeutic options. Therefore, identification of new clinically relevant targets and biomarkers represent an urgent need for these patients [Chemotherapy, with or without the addition of Bevacizumab, remains the standard treatment for RASpatients –4.MUT tumors [MUT-driven tumors [Recent studies have tested the antitumor activity of the combination of anti-cell cycle drugs and inhibition of the Ras pathway, in RAST tumors –15. In tT tumors . CDK4/6in tumors , 17–19. Kip1 (hereafter p27) is a cell cycle inhibitor, member of the CIP/KIP family of CDK inhibitors. p27 is an haploinsufficient oncosuppressor and its functions can be both CDK-dependent and -independent [CDKN1B gene, encoding for p27, is rarely due to mutation or deletion [e.g. MAPK, PI3K) that lead to p27 protein phosphorylation and subsequent degradation or cytoplasmic delocalization [p27ependent , 21. In deletion , 23. Condeletion , 21. p27deletion –27. The lization . Also nolization , 29–31. lization , 29, 30.lization .MUT lung tumor cells demonstrated that prolonged exposure to Palbociclib induced an increased expression of p27 that was then able to form an active complex with cyclin D1 and CDK6, contributing to the resistant phenotype [A complex regulatory loop exists between Ras and p27 to control cell cycle progression in response to mitogenic stimuli , 33–35. henotype .MUT CRC, is still unexplored. Here, we test this possibility and investigate the role of p27 in RASMUT CRC response to Palbociclib.Whether these mechanisms may be working also in vivo and, particularly, in RASn = 201) were retrospectively collected from patients with primary and metastatic colorectal cancer upon signing a written informed consent, in accordance with recognized ethical guidelines and followed by approval of University of Rome “Sapienza” Santo Andrea Hospital .Formalin-fixed and paraffin-embedded (FFPE) specimens  supplemented with 10% FBS (Carlo Erba) and 1% PS (Lonza).All colorectal cancer cell lines were maintained in RPMI 1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum  and 1% penicillin and streptomycin . All cell lines have been authenticated using the Cell ID TM System (Promega). SW480 Palbociclib-resistant cells were generated by continuous treatment with increasing doses (from 0.25 to 2 µM) of Palbociclib  . Kill cuRNA extraction, qRT-PCR, protein lysates, immunoprecipitation (IP), Western blot analyses and in vitro kinase assays were performed as already described , 39, 40.Animal experimentation was reviewed and approved by the Centro di Riferimento Oncologico di Aviano (CRO) Institutional Animal Care and Use Committee (OPBA). All animal experiments were conducted in adherence to international and institutional committee ethical guidelines. NOD.CB17-PrkdcSCID (NSG) male mice (Charles River Laboratories) were orthotopically injected with HCT-116-Luc2  cells and treated with Palbo and Saracatinib, alone or in combination, as described in Supplementary Information. Combined macroscopic and pathological analyses of the mouse organs were performed as described in .p < 0.05. Detailed methods can be found in Supplementary Information.Statistical significance, averages, median, and SD were determined by using GraphPad PRISM software (version 6.01), using the most appropriate test, as specified in each figure. Significance is indicated by a CDKN1B, is rarely mutated in cancer and its regulation mainly occurs at post-transcriptional level [BRAF (BRAFMUT) or RAS mutation   Fig. . CRC witors Fig. . AltogetBRAF or RAS  or Caco-2 (KRASWT/BRAFWT) cells did not modify Palbo-sensitivity  was overexpressed , compared to low p27 ones  or with stathmin (p271-170), a cytosolic protein involved in the regulation of microtubule stability and vesicular trafficking and previously shown to interact with p27  overexpressing HCT-116 KRASMUT cells, silenced for p27 (sh-p27) or control (sh-NT) , by Brk or Src family members, can allosterically activate CDK4-Cyclin D complexes , 30, 43 MUT cells. p27 was tyrosine phosphorylated in parental CRC cells and this phosphorylation decreased under Palbo treatment, both in basal condition and after serum stimulation , in order to see its effect in combination with Palbo. It is to mention that we observed some toxicity in Saracatinib treatment arms, which was quite unexpected since its use in patients was reported to be well tolerated [WT HCT-116 cells were quite resistant to Palbo treatment, but the combination with Saracatinib significantly reduced disease dissemination, with visible, although not significant, stage reduction , KRASMUT CRC displayed higher levels of active Src, compared to KRASWT, either BRAFMUT or BRAFWT , it is much less frequent in breast cancer and, in particular, in ER positive breast cancers (less than 1%), the tumor subtype in which CDK4/6i are more active [Here, we investigated the mechanism linking p27 expression and Palbo-response in CRC cells and showed that high p27 expression decreases the activity of Palbo in KRASgression , 46. Howgression . Importagression , 43 and gression . Overallgression . In thise active . Similare active , in breae active .MUT and not in BRAFMUT cells. Since these proteins belong to the same pathway one could expect that p27 exerted similar function(s) in both contexts. However, this is not very surprising since it is well known that KRASMUT and BRAFMUT identify two quite different molecular subtypes of CRC [MUT, but not BRAFMUT, has the ability to co-activate the Src pathway, as already demonstrated in pancreatic cancer [MUT, correlating with tumor dissemination and poor prognosis [MUT CRC, a possibility that will certainly need further investigation and validation in larger cohorts of human samples and patients.An intriguing observation resulting from our work, for which we do not have a full explanation yet, is that p27 confers resistance to Palbo only in KRASs of CRC . We can c cancer . This hyrognosis , 52. Sine.g. NCT03981614, NCT02703571, NCT04165031, NCT02745769). Notably, the JUNIPER trial in KRASMUT lung cancer has showed that the CDK4/6i Abemaciclib performed better than the EGFR inhibitor Erlotinib, in term of both objective responses and progression free survival (NCT02152631).The addition of CDK4/6i to endocrine therapy in breast cancer patients has led to significant improvement of progression-free survival. Based on these promising results, great hopes are now pinned on the use of CDK4/6i in different cancers types, especially when used in combination with other targeted therapies or chemotherapy . AccordiMUT-driven tumors and, therefore, predictive markers of response to select patients and ways to circumvent de novo or acquired resistance are greatly needed. In this context, our work that demonstrates in KRASMUT CRC that low p27 expression identifies high-risk patients that could benefit from the use of CDK4/6i, while high p27 expression, coupled with tyrosine phosphorylation by Src, may lead to Palbo-resistance, could be of great help in defining the clinical treatment decision tree for these patients. Our data further indicate that Src inhibition might increase Palbo activity in high-p27 expressing tumors and prevent the appearance of p27-induced CDK4/6i-resistance. Notably, Saracatinib has shown little efficacy as a single agent in previously treated metastatic colorectal cancer patients, but it is known for its high tolerability [These data support the possibility that the use of CDK4/6i might be more successful in a subset of KRASrability , 45. TheMUT CRC Palbo-resistance could be overcome by the concomitant use of Src inhibitors and that evaluation of p27 expression in tumor tissue might be sufficient to select KRASMUT CRC patients who may benefit from administration of CDK4/6 inhibitors, alone or in combination with Src inhibitors.Overall, our findings indicate that in the context of KRASSupplementary Information


Malignant T cells may overexpress programmed death ligand 1 (PD-L1), which signals via programmed death-1 (PD-1) receptor, and provides an inhibitory signal on normal T-cells further suppressing antitumor immunity. They can also express PD1, which may act as a tumor suppressor on malignant T cells.The NIVEAU trial is an ongoing international, multicenter, randomized, open label, phase 3 study testing Nivolumab (Nivo) in combination with Gemcitabine and Oxaliplatin (Gem-Ox) [± Rituximab] for patients with aggressive (B and T cells) non-Hodgkin lymphoma, in first relapse or progression, who are not eligible for high-dose chemotherapy (NCT03366272). Here, we performed a preliminary analysis of the experimental arm (Nivo-GemOx) of the PTCL cohort to assess the safety and efficacy of this regimen in this population.2 at day 1) and Oxaliplatin (100 mg/m2 at day 1) in 2-week intervals followed by additional 18 infusions of Nivolumab (3 mg/kg) biweekly as consolidation over a period of 1 year or until progression. Response was evaluated after 4 and 8 cycles of Nivo-GemOx according to the Lugano classification,5 and every 3 months by CT-scan during consolidation therapy. Each progression/relapse of PTCL had to be reported as an SAE.Key eligibility criteria include the following: first relapse or progression of PTCL, ineligibility for high-dose therapy (defined as age > 65 years or older than 18 years if HCT-CI score > 2), only 1 prior chemotherapy regimen including an anthracycline. Patients in the experimental arm were planned to receive 8 cycles Nivolumab (3 mg/kg at day 0 of cycle 1 and day 1 of cycle 2–8) plus Gemcitabine (1000 mg/mhttp://links.lww.com/HS/A216): 4 (33%) PTCL NOS, 3 (25%) AITL, 1 (8%) nodal TFH-PTCL, 2 (17%) ALCL ALK-, 1 (8%) EATL, and 1 (8%) MEITL. Median age was 69.5 years , 7 (58%) patients were male, 2 (17%) had received a prior autologous stem cell transplantation, and 5 (42%) were refractory to first line therapy. At enrollment, performance status was 0–1 in 9 (75%) pts and 2 in 3 (25%) pts, 11 (92%) had Ann Arbor stage III–IV, 2 (17%) had B-symptoms, 7 (58%) had more than 1 extranodal site and 4 (33%) had elevated LDH. PD1 and PD-L1 were expressed by the tumor cells in 6/10 (60%) and 2/11 (18%) patients, respectively  included 12 patients enrolled in the experimental arm .Patients have received a median of 6  cycles of GemOx and 8  infusions of nivolumab. Treatment was prematurely discontinued in 9 patients (7 during induction and 2 during consolidation), due to lymphoma progression (n = 6), toxicity (n = 2), or intercurrent disease . There were 28 SAE in 11 patients, including 8 progressive diseases . Unfortunately, the reference period cannot be a wash-out period (off-therapy) in aggressive lymphoma. In this fast-growing disease, the reference period is the prior line of therapy, as only on-treatment CT-scans are available before initiation of the experimental salvage treatment regimen. Thus, the standard criteria for hyperprogression, which have been established in solid tumors, could not be strictly applied in our cohort of patients.6 Nevertheless, patient 9 , which progressed after first-line therapy of 2 cycles of CHOEP and 2 cycles of high-dose Methotrexate experienced a second progression after the second cycle of GemOx. Patient 12 , which progressed 1 month after 6 cycles of CHOP plus additive radiotherapy experienced a rapid second progression within its first cycle of study treatment, when again a partial remission lasting several months could be achieved after switching to third-line therapy. In this patient, nivolumab might have promoted lymphoma progression. However, shorter duration of remission in relapse represents a common phenomenon in lymphoma. Thus, establishing scientific criteria for hyperprogression are warranted. We did not find a clear correlation between PD1/PD-L1 expression on tumor cells and response to Nivo-GemOx. However, PD-L1 may also be expressed by bystander cells from the tumor microenvironment. This will be further explored in the final analysis of the clinical trial. Among the 9 responding patients, the median DOR was 14.9 months. Median PFS2 (time from randomization to second relapse/progression/death) was 6.9 months  versus 7.7 months  for PFS1 (time from diagnosis to first relapse/progression). Importantly, PFS2 was superior to PFS1 in 4 out of 10 patients (40%), and not informative in 2 pts: patient 10 who is still on therapy (ongoing PFS) and patient 8 who died prematurely of infection  patients achieved an objective response . Two patients experienced primary progression upon Nivo-GemOx Table : patient8 other studies demonstrated that anti-PD1 had efficacy in these neoplasms.11 Interestingly, the combination of Nivolumab with GemOx lead to high response rates and prolonged remissions (compared to first-line treatment) in a subset of patients. Longer PFS after salvage therapy suggests that it is more effective than the previous line of therapy, a CHOP-based regimen in our study. The combination of anti-PD1 antibody with chemotherapy may indeed have additive or synergistic clinical activity. Chemotherapy, notably gemcitabine and oxaliplatin, can promote tumor immunity by inducing immunogenic cell death and by disrupting the immunosuppressive tumor microenvironment.12 Additionally, anti-PD1 therapy may not only stimulate the antitumor immune response but also may sensitize to chemotherapy.15 In non-Hodgkin lymphoma, a retrospective study showed that the duration of response (DOR) to therapies given after checkpoint blockade therapy were longer than the DOR to treatment immediately prior to checkpoint blockade therapy, suggesting a potentiation effect of the immunotherapy.14 Cytostatic drugs like gemcitabine and oxaliplatin are disrupting DNA replication, which requires transition into cell cycle.17 One might speculate, that blocking the PD1 pathway results in increased T-cell receptor signaling and proliferation, rendering the cell more susceptible for chemotherapy.1These preliminary results show that nivolumab in combination with GemOx is well tolerated. Although some reports raised the concern that anti-PD1 therapy may promote tumor proliferation in T-cell lymphoma,Pdcd1 genomic deletions) will also be performed to identify predictive markers of efficacy. The NIVEAU phase 3 trial is actively enrolling patients.Overall, these preliminary results show encouraging efficacy and safety profiles of the Nivo-GemOx regimen in R/R PTCL. These findings will have to be confirmed on a larger number of patients and by comparing this combination with the control arm (Gem-Ox) once the NIVEAU study will be completed. Translational research  Abbvie, Bayer, Celgene, Janssen, Roche; speakers honoraria from Bayer, Celgene, Gilead, Janssen, Roche; scientific advisory board from Astra Zeneca, Bayer, Beigene, Celgene, Gilead, Janssen, Novartis, Roche. H.T. received honoraria from Roche, Karyopharm, Aatra-Zeneca, Servier, Janssen-Cilag, BMS. S.M. did travel grants from Amgen, Abbvie; honoraria from Amgen, Novartis, Roche. G.C. received consultancy from Roche, Celgene; honorarium from Sanofi, Abbvie, Jansen, Gilead, Roche, Celgene. A.K. received travel grants from Roche and honoraria from Amgen, Novartis, BMS, and Takeda. T.W. received travel grants from Gilead, Roche, Takeda, honoraria from Roche and Takeda, research funding from Riemser and Takeda. D.H. received honoraria from Bayer Vital GmbH. C.H. received honoraria from Roche, Janssen-Cilag, Gilead, Takeda, Miltenyi and Servier and travel grants from Amgen and Celgene. P.G. received consultancy from Takeda, Gilead; research funding from Takeda, Innate Pharma; Sanofi Honoraria from Takeda; travel grants from Roche. G.H. received consultancy from Roche, BMS, MSD; research funding from BMS, Roche, Acrotech, Spectrum, Amgen; and travel grants from BMS, Roche. The NIVEAU is an ISR, which is financially supported by Bristol Myers Squibb with no influence on the content. All the other authors have no conflicts of interest to disclose.


Notably, the organ deposition patterns of those transplutonium actinides were element-dependent, particularly in the liver and skeleton, where lower atomic number radionuclides showed up to 7-fold larger liver/skeleton accumulation ratios. Nevertheless, the metal content in multiple organs was significantly decreased for all tested actinides, particularly in the liver, after administering the therapeutic agent 3,4,3-LI post-contamination. Lastly, the systematic comparison of the radionuclide biodistributions showed discernibly element-dependent organ depositions, which may provide insights into design rules for new bio-inspired chelating systems with high sequestration and separation performance.Transplutonium actinides are among the heaviest elements whose macroscale chemical properties can be experimentally tested. Being scarce and hazardous, their chemistry is rather unexplored, and they have traditionally been considered a rather homogeneous group, with most of their characteristics extrapolated from lanthanide surrogates. Newly emerged applications for these elements, combined with their persistent presence in nuclear waste, however, call for a better understanding of their behavior in complex living systems. In this work, we explored the biodistribution and excretion profiles of four transplutonium actinides ( Transplutonium actinides are among the heaviest elements whose macroscale chemical properties can be experimentally tested. Food and Drug Administration (FDA) approved calcium and zinc salts of diethylenetriamine pentaacetic acid (Ca-DTPA and Zn-DTPA) to treat internal contamination with Pu, Am and Cm.14 Even though DTPA is the first drug approved to treat this type of radiological contamination, its therapeutic performance is hampered by multiple factors, including (1) a need to administer it in large quantities,15 (2) its competition with biological ligands  for the binding of the metal,16 and (3) its inability to remove radionuclides deposited in organs.17The transplutonium actinides Cm, Bk, Cf and Es are among the heaviest elements whose macroscale chemistry can be experimentally tested.18 This synthetic chelating molecule shows high binding affinity for actinides and lanthanides,19,20 selectivity for f-block elements over biologically-relevant cations,21,22 formation of excretable complexes with these elements,23 and biocompatibility at therapeutic dosages.23In vivo studies have demonstrated the superior decorporation performance of 3,4,3-LI for multiple elements, including U(vi), Np(v), and Pu(iv), when compared to DTPA and other chelating agents.23–25 As a result, 3,4,3-LI has been approved for Phase 1 first-in-human clinical studies, as a decorporation agent for actinides.18 The aforementioned in vivo studies, however, focused on lanthanides and earlier actinides, and recent spectroscopic and separation studies with Cm, Bk and Cf revealed coordination chemistry differences,19,26,27 prompting us to question whether such effects could translate to different biodistribution patterns and if 3,4,3-LI would preserve its decorporation performance amongst heavier radionuclides.In order to overcome the limitations of DTPA salts as decorporation agents, the octadentate hydroxypyridinone-based chelator 3,4,3-LI was developed.248Cm, 249Bk, 249Cf and 253Es. Although the main two deposition regions, skeleton and liver, were the same for the four metals, the total organ retention and evolution over time was element-dependent, with lower atomic number radionuclides showing higher liver and lower skeleton accumulations. Prompt post-contamination decorporation treatments with DTPA and 3,4,3-LI   resulted2.2.1.248Cm, 249Bk, 249Cf, and 253Es were chosen for their availability and because their half-lives  were long enough to perform the experiments. The radionuclides were injected intravenously as citrate solutions with final administered activities for each actinide ranging between 0.23 and 0.93 kBq per mouse , metal-binding proteins, such as hemoglobin, transferrin, and fetuin, are in the micromolar and millimolar range in mouse blood (Table S2−1 per mouse) through intraperitoneal injection 1 h after contamination, an administration time widely used to assess decorporation treatments,18 and were euthanized 24 h after actinide exposure. All mice showed steady body weight, and no visible or palpable dermal infections, impaired mobility, or ascites were detected during the study, indicating a lack of acute toxicity.The biodistribution of transplutonium elements was tested in young adult female Swiss-Webster mice.  Table S1. These a Table S2. It is w Table S2. However248Cm accumulation in the bones was steady (between 28 ± 2 and 24 ± 4% RD) over 24 h, while 249Bk, 249Cf and 253Es content increased from around 32% up to 42 ± 3, 50 ± 2, and 49 ± 2% RD, respectively. The different behaviours between the four isotopes were more pronounced in the liver, the organ with the second largest actinide content  and 253Es (12 ± 1% RD) after 24 h. The higher liver accumulation by earlier transplutonium actinides is consistent with a past study with trivalent 241Am (0.9 kBq per mouse), which showed 49% RD liver content after 24 h.18 The experimental protocol used in ref. 18 is the same as that used in our current study. Regarding other organs, kidneys  are poorly recognized by the transferrin receptor, and only one isoform containing one Pu4+ ion and one Fe3+ ion is actively recognized and endocytosed.37 Hence, Pu4+ is only moderately internalized by hepatic cells compared to other metals.38 Thus, the direct affinities between the metals and the transferrin may not be as important on defining the actinide liver uptakes, as the interactions between the metal complexes and the transferrin receptor are. There is not a systematic study that explores the binding between trivalent actinide–transferrin complexes and the receptor. However, a study with lanthanides indicated decreasing affinity of the receptor for the metal–transferrin complexes with increasing atomic number (La3+ ∼ Nd3+ > Gd3+ > Yb3+),39 which, if trivalent actinides follow the same trend, may explain the larger liver deposition of early transplutonium elements that we observed. It is worth noting that the burden transition from a higher liver accumulation to a higher skeleton accumulation occurs between Cm3+ and Bk3+ for the actinides  or the actinide transition would appear around Cf3+ (with an ionic radius intermediate between those of Sm3+ and Eu3+).40 This transition, however, could be explained based on differences on covalency between the lanthanide and actinide series. Over the past decade, a combination of experimental and theoretical studies has evidenced increasing covalent character in bonding across the late actinide series, contrasting with the trivalent lanthanides.41–43 In addition, small molecular transplutonium complexes have been shown to display strong energy degeneracy-driven covalency, distinct from the more traditional overlap-driven covalency typical of the early actinides.44–46 Those are very subtle differences but they could still affect several features, including bond lengths and electron density around the metal center, metal binding kinetics, or metal complex thermodynamic stability and could potentially explain variations in 4f- or 5f-element binding by different biological molecules, such as bone glycoproteins, small molecular ligands circulating in the blood or common metalloproteins.Regarding liver accumulation, actinides interact with multiple metal-binding proteins in the liver uptake pathway, including transferrin, ferritin, and calmodulin.ctinides  and betw47Lastly, the absorbed dose rates for each radionuclide in the liver and skeleton (the main two deposition regions) were calculated at 24 h to further confirm that the injected doses were low enough to avoid any acute radiation damage. The absorbed dose rates were sub-μGy/s for all actinides Table S3, below t2.2.248Cm, 249Bk, 249Cf, and 253Es to 57 ± 3, 53 ± 6, 47 ± 5, and 48 ± 7% RD, respectively  significantly prevented actinide accumulation in liver despite the 1 h delayed treatment is the chelator's fast biodistribution into this organ, which occurs within 5 min after injection.49Mice that were treated with chelating agents received the treatment 1 h after contamination, and were euthanized 24 h post-metal exposure. Administration of DTPA reduced the total retained ectively . The bod2.3.24 Noteworthy, we observed significant differences and opposite trends in ligand-promoted excretion as a function of the metal, following the order 248Cm < 249Bk < 249Cf ∼ 253Es for DTPA and 253Es < 249Cf ∼ 249Bk < 248Cm for 3,4,3-LI. The trend seen with DTPA correlates with increasing stability constants for complexes formed from Cm to Es , which is not the case for DTPA.In addition to the bioaccumulation studies, the mice excreta were also collected and radioanalyzed.  Table S4, with hi2.4. in each organ and excreta tested, for the control, HOPO treatment, and DTPA treatment groups, 1 h post-contamination  output of 241Am is smaller  than that of 248Cm in control contaminated animals, that difference is much increased after one 3,4,3-LI chelation treatment, as depicted by the much brighter colours in the upper right corner of the top right panel of 50 or for the large-scale extraction of these elements from diluted environments such as seawater,51 many biological and biologically-inspired molecules have recently emerged as potentially promising systems for f-element separations. Current state-of-the-art in this field runs the gamut from biopolymers, organic acids, as well as small molecule metallophores, peptides and proteins produced by microbial species.52 However, mammalian systems have not yet been explored in that context. The results presented here indicate that the molecular mechanisms involved in the mammalian transport and storage of actinide contaminants could eventually be decrypted, improved, and utilized to discriminate f-block metals. One may even envision the engineering of new bioreactors, such as modified spheroid reservoir bioartificial livers,53 that would leverage these mechanisms.To further provide a thorough perspective of the different actinide bioaccumulation profiles as well as their respective variations after chelator administration, we systematically compared deposition ratios of the tested radionuclides mination . In this3.248Cm, 249Bk, 249Cf and 253Es, in a live mouse model. Their deposition in organs was observed within the first five minutes after contamination, and skeleton and liver were the main accumulation regions. Despite their similar charge and ionic radius, the distribution of radionuclides was element dependent, where lower atomic number transplutonium elements showed up to 7-fold higher liver/skeleton deposition ratios compared to the heavier tested actinides. Treatment with both DTPA and 3,4,3-LI 1 h after contamination promoted higher radioisotope excretion, with the latter showing better performance (around 3-fold higher clearance compared to control). Although mice do not use actinides in their metabolism, transplutonium element interaction with endogenous chelators seems to be element dependent, resulting in different in vivo behaviour. Such discernible differences in a complex multi-component living system may provide future insights for developing new bio-inspired strategies for efficient sequestration and separation of transplutonium elements.In summary, we studied the biodistribution and excretion of the heaviest transplutonium elements available, namely in vivo experiments. R. M. P., D. D. A., G. J.-P. D., and R. J. A. analyzed the data. All authors discussed the experimental results and contributed to the manuscript.D. D. A., G. J.-P. D., B. K., and R. J. A. designed the research. G. J.-P. D. and R. J. A. prepared radioisotope solutions for experimental procedures. D. D. A., B. K., S. S. G., and E. E. J. performed R. J. A. and G. J.-P. D. are listed as inventors on patent applications filed by the Lawrence Berkeley National Laboratory (LBNL) and describing inventions related to the research results presented here. The authors declare no competing financial interest.SC-012-D0SC06610A-s001


The outcome of COVID-19 appears to be influenced by vitamin D status of population. Although epidemiological data indicate that COVID-19 produces more severe symptoms and higher mortality in elderly in comparison to young patients and in men in comparison to women to date sex and age differences in vitamin D status in infected patients have not been evaluated yet. In this study we evaluated the levels of circulating 25(OH)D in patients hospitalized for COVID-19 divided accordingly to their sex and age. We also correlated 25(OH)D levels with patient’s respiratory status  and with sex hormones plasma levels to analyze the potential relationship of these parameters. We found no significant differences in plasma levels of 25(OH)D between pre- and post-menopausal female patients and age matched male patients. Interestingly, the 25(OH)D plasma levels positively correlated to PaO2/FiO2 ratio only in young patients, regardless of their sex. We also found a significantly positive correlation between 17β-estradiol and 25(OH)D in elderly women and between testosterone and 25(OH)D in elderly men, supporting the role of sex hormones in maintaining 25(OH)D levels. In conclusion, we suggest that a synergy between vitamin D and sex hormones could contribute to the age-related outcome of COVID-19. The outcome of coronavirus disease 2019 (COVID-19) appears to be influenced by the interaction among genetic, hormonal and environmental factors. In this context, low levels of circulating 25-hydroxyvitamin D [25(OH)D], the biomarker of vitamin D status, represent a risk factor for COVID-19 and 1,25(OH)2 vitamin D(3) (the active metabolite of vitamin D) seems to play a protective role in this disease by controlling the cytokine storm, enhancing the production of antimicrobial peptides and maintaining the integrity of the epithelium . MoreoveU test and Spearman’s rank correlation using GraphPad Prism . A p value <0.05 was considered statistically significant.To address this issue, we measured plasma levels of 25(OH)D, testosterone and 17β-estradiol from 160 adult patients  admitted to Lazzaro Spallanzani Hospital, Rome, Italy between March and September 2020, positive for SARS-CoV-2 by RT–PCR from nasopharyngeal swabs, and able to provide informed consent. Plasma samples were collected at clinical admission. Partial pressure of oxygen/inspired oxygen concentration ratio (PaO2/FiO2 ratio), representing a valuable clinical measure of the patient’s respiratory status, were recorded during clinical admission. ELISA kits for 25(OH)D  testosterone  and 17β-estradiol  were used according to manufactures’ instructions. Statistical analysis was performed by the Mann–Whitney p = 0,03 and p = 0,01 respectively, p = 0.01, p = 0.04, No significant differences in plasma levels of 25(OH)D were detected between female and male patients and among the four subgroups of patients divided according to sex and age . InteresIn this study we analyzed the 25(OH)D plasma levels in patients with COVID-19 divided accordingly to their sex and age and we found a positive correlation between vitamin D status and respiratory functionality only in young patients, regardless of their sex. This result was consistent to previous studies on the association of serum 25(OH)D concentration with the improved lung function, in both females and males . We askeIn conclusion, it is tempting to hypothesize that the synergy between vitamin D and sex hormones could contribute to the age-related outcome of COVID-19. Controlled studies are needed to determine the role of vitamin D supplementation in combination with estrogens or androgens in improving respiratory function of female and male patients, respectively.All in all, the collection, analysis and report of data on COVID-19 disaggregated by sex and age seem to be a duty to achieve the appropriateness of the prevention, treatment and control of the disease.Simona Anticoli, Maria Bellenghi, Veronica Bordoni, Marta Camici1, Rita Casetti, Carlotta Cerva, Pierangelo Chinello, Eleonora Cimini, Davide Roberto Donno, Maria Luisa Dupuis, Francesca Faraglia, Katia Fecchi, Roberta Gagliardini, Germana Grassi, Rachele Di Lorenzo, Manuela Macchione, Gaetano Maffongelli, Gianfranco Mattia, Eugenia Milozzi, Silvia Mosti, Giada Pontecorvi, Rossella Puglisi, Alessandra Sacchi, Eleonora Tartaglia, Serena Vita.


The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes  exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells . Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration. Thus, motility has been studied in models with different levels of microenvironment complexity , topographies (that do or do not impose cellular deformation), or that mimic their transmigration through tissue layers  influx, and metabotropic (P2Y) receptors that trigger Ca2+ release from intracellular stores  and pannexins (PANXs), and its coupling to purinergic signaling represents a widely used mechanism for cell communication, which plays a role in paracrine (between cells) and autocrine (cell autonomous) signaling (P2 receptors are classified into ionotropic (P2X) receptors that allow calcium ions  and ions between the cytoplasm and the extracellular milieu . Upon dovia protein-protein interaction with a microtubule stabilizer  proteolytic activity, preventing extracellular matrix modification . Therefo section Table 11 receptors by extracellular ATP, released via PANX1 channels  neutrophils leave the bloodstream to invade vascular tissues . In the channels .2 2  gradient, which mimics a bacterial-induced chemotactic response, also depends on extracellular ATP sensing. During FMLP-induced migration ATP is released in a polarized manner, as PANX1 is polarized to the leading edge together with F-actin . This po sensing , 54. Int sensing . The con sensing . Since a1 receptors, this reduces the migration of human neutrophils and HL-60 neutrophil-like in an under agarose assay, and then acts as a stop signal  maxi anion channels , 42, butp signal .Overall, PANX1 contributes to neutrophil cell migration by establishing a polarity axis, which is supported by re-localization of the actin cytoskeleton, and the purinergic signaling-related proteins involved in the migratory response. However, the putative role of PANX2 and PANX3, and in pathological conditions remains largely unexplored.7 receptor modulates the balance between the number of differentiated Th17 and Treg lymphocytes  search in the lymph nodes, T cells have a diffusive and random migration (Brownian type), whereas less diffusive chemotactic movements (Lévy type) is exhibited by recently activated T cell to migrate into secondary lymphoid organs for priming; or highly directional migration  induced by haptotaxis cues and chemotaxis gradient caused by cognate APC at the peripheral tissues . Adaptivphocytes . Accordiphocytes –65. In b+ immature T cells  stimulation induces PANX1 channel opening, cell polarization, and migration of CD4 T cells , 48, as  T cells . In thistivation Figure 1tivation . Thus, ptivation , suggesttivation . Interestivation .+ and CD8+ cells, PANX1 is abundantly present at the plasma membrane, whereas PANX2 abundance is much lower  activation and F-actin cytoskeleton to the cell rear  normally reside in peripheral tissues where they scan the microenvironment in search of danger signals , 70. Afty phases , 32, 71.ell rear Figure 1igration . PANX1 aigration , its actigration , 74. ThuActivating signals trigger a maturation program in which several phenotypic changes, including the decrease of macropinocytosis, are required for the fast migration of DCs , 69. IntThese data suggest that PANX1 channels might contribute to the early stages of DC migration upon danger signal detection, and that later stages including CCR7-dependent chemotaxis relies in the activity of CXs. This putative functional distinction between CXs and PANXs during DC migration might rely in different protein turnover at the plasma membrane, and/or the non-channel signaling function, as is shown for CX43 .Dendritic Cells, DCs). However, the molecular mechanism for ATP release was not elucidated. Monocytes, and macrophages, express both CXs and PANXs, but only CXs role has been shown during extravasation and migration. Particularly, CXs contribute by forming gap junction channels between monocytes and the endothelium  or monocyte-derived DCs (Mo-DCs) upon sensing of danger signals at the injury site . During othelium , 40, 78.othelium , remainsin vivo, which extend their processes towards the injury site, a response that might require PANX1 channel activation . However, this response might be specific different macrophage subsets, as PANX1 channels have only a mild contribution to C5a-induced chemotaxis in peritoneal macrophages, which was dependent on P2Y2 and P2Y12 receptors , macrophages polarize to M1 or M2 macrophages, which exhibited different migratory properties . InteresImmune cell migration is not only induced by activation of PANX expressed in the migrating cell, but also can be indirectly promoted by PANX channels activated in the tissue. Initial observations by Chekeni et al, revealed that PANX1 channels were required for the release of “find me” signals  during T cell apoptosis, which triggered monocyte recruitment .in vitro experiments with a muscle cell line that form myotubes show that treatment with palmitate induces ATP release from myotubes, which triggers monocyte recruitment . Exposure to lipoapoptotic supernants elicits monocyte recruitment in an ATP-dependent chemokine-independent manner . PANX1 cruitment . Interesruitment . Consequruitment . A similruitment . PANX3 Kruitment .via PANX1 channels from the epithelium of the choroid plexus, although epiplexus do not express PANX1   , 114. Th1 (CD90) , 116. In+ influx , 118, re7 receptors allowing the establishment of molecular toolkit required for Thy-1 signaling  model hSOD1G93A transgenic mice present an increased abundance of several migration-related molecules, including αvβ3 Integrin, syndecan-4 proteoglycan, P2Xand CX43 . Thy-1 rand CX43 . Intriguin vivo)  , 120. Inin vivo) . In the in vivo) . Howeverin vivo) , 103. Thin vivo) . Interesin vivo) . Altogetin vivo) Figure 3in vivo invasion in melanoma, breast and testicular cancer cells: B16-BL6, B16-F10; CN34, CN-LM1A, MDA-MB-231 MDA-MB-468, MDA-LM2, and I-10 cells, respectively , MDA-MB-231 cells are sensitive to Thy-1 that increases cell migration by releasing ATP ack loop . Consequack loop , suggestWhich are the signals that lead to a positive or negative modulation of PANX1 on migration? As reviewed in this section, we hypothesize that it might depend on the cell type Table 2,+ and CD4+ T cell from old mice present lower migration and microtubules gene expression , which are master regulators of cell migration. There is evidence of direct and indirect modulation of PANX channels by the cytoskeleton, which modifies membrane dynamics . In addie CaMKII , or by ae CaMKII , 148 , MINECO Spain PID2020-116086RB-I00 (PS), Forschungszentrum Medizintechnik Hamburg (FMTHH) 04fmthh2021 (PS), ANID Postdoctoral Project N° 3200342 (PH), MILENIO ICM-ANID P09-022-F (AP and PH).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.