The reduction in neuroinflammation after 4D-sEVs delivery to the injury site epicenter led to significant neuroprotection, as evidenced by the number of surviving spinal neurons. Therefore, applying this novel 4D culture-derived Small Extracellular Vesicles could effectively curb the inflammatory response and increase tissue repair after SCI.Effectively reducing the inflammatory response after spinal cord injury (SCI) is a challenging clinical problem and the subject of active investigation. This study employed a porous scaffold-based three dimensional long-term culture technique to obtain human umbilical cord mesenchymal stem cell (hUC-MSC)-derived Small Extracellular Vesicles (sEVs) . Moreover, the vesicle size, number, and inner protein concentrations of the MSC 4D-sEVs contained altered protein profiles compared with those derived from 2D culture conditions. A proteomics analysis suggested broad changes, especially significant upregulation of Epidermal Growth Factors Receptor (EGFR) and Insulin-like Growth Factor Binding Protein 2 (IGFBP2) in 4D-sEVs compared with 2D-sEVs. The endocytosis of 4D-sEVs allowed for the binding of EGFR and IGFBP2, leading to downstream STAT3 phosphorylation and IL-10 secretion and effective induction of macrophages/microglia polarization from the pro-inflammatory M1 to anti-inflammatory M2 phenotype, both  Spinal cord injury (SCI) is detrimental to the central nervous system and often results in lifelong sensory and motor impairments . The hosPrevious studies have developed a three dimensional (3D) culture methodology for constructing neural tissue or spinal cord-like tissue derived from adult stem cells in a trophic factor-defined 3D scaffold. With the dynamic interactions between stem cells and the ambient 3D microenvironment over time, stem cells can recapitulate developmental scenarios and develop into homeostatic entities resembling critical tissue features such as functional cells and their extracellular matrix (ECM). For example, seeding neural stem cells (NSCs) in a sustained neural trophic factor (NT)-3 releasing bioscaffold resulted in the formation of a self-organized neural network tissue with mature neuronal function . This prIn this study, for the first time we characterized the main features of sEVs derived from the 4D-culture of MSCs  and investigated their effect on regulating the inflammatory response in the injured area of the spinal cord. The results suggested a potent anti-inflammatory effect of the 4D-sEVs. This feature may enable 4D-sEVs use in future clinical applications for SCI patients.5 hUC-MSCs, and ten scaffolds/well were cultured in a six-well plate (3D-scaffold group). Ten cell-loading scaffolds were cultured in 10 mL culture medium in each well of a six-well plate. Equal numbers of hUC-MSCs were plated in cell culture flasks (this is the 2D group). The two groups were incubated at 37°C for 14 days, and the culture medium was changed every 2 days. After 14 days, the growth medium was removed, cells were washed 3 times with D-hank’s, and then serum-free hUC-MSC sEVs secretion-promoting medium Ultra CULTURETM  was added to the cells, and the supernatant was collected after 48 h.As previously described , a threeg for 10 min, and then the supernatant was collected and filtered using a 0.22 μm filter. Centrifugation was performed at 2000×g for 10 min and 10,000×g for 30 min to remove cell debris. Next, We used overspeed centrifuge  with the rotor of type 70 Ti and the k-factor of 44 to extract extracellular vesicles. Centrifugation was performed at 100,000×g (37000 RPM) for 90 min, the supernatant was removed, and the pellet was resuspended in PBS. Finally, centrifugation was performed again at 100,000×g for 90 min, and the pellet was resuspended in 200ul PBS and then stored at −80°C.The collected sEVs were purified using centrifugation, adapted from a previous protocol . The supThe yield of sEVs/per cell was calculate by dividing the number of sEVs by the total cell number of MSCs within the scaffolds following 4D culturing. The cell number in each scaffold was numerated after detaching them with enzymes .The morphology of the sEVs was determined by Transmission Electron Microscopy (TEM). Fifty (50) μg of sEVs were diluted (1:40) with PBS, then diluted sEVs suspension (20 μL) was dropped on a 300-mesh cell strainer for 10 min. Excess liquid was absorbed with filter paper, counterstained with 1% phosphotungstic acid for 30 s, and observed under a transmission electron microscope.7–109 and the maximum diameter of 2 um to detect the particle number and size distribution of sEVs. Samples were diluted  with PBS, injected into the device manually, we set the threshold at 5 and videos were acquired at ambient temperature (camera level 9) for 1 min per sample.We used Nanoparticle Tracking Analysis  with the particle concentration of 10sEVs were diluted with PBS at 1:10, 1:50, 1:100, and 1:200, respectively. The protein standards and sEVs samples were added to a 96-well plate according to the BCA Protein Concentration Assay Kit instructions , and the absorbance was measured at a wavelength of 562 nm to detect protein concentration of sEVs. Therefore, the protein concentration for each particle could be calculated by dividing the total protein concentration by the particle number of each sample.Under sterile conditions, the sEVs (100 μg) were resuspended in PBS (1 mL), and 10 µL Dil Cell-Labeling Solution  was added to the sEVs suspension. After gently blending, the suspension was incubated at 37°C for 10 min. Then, the dye-labeled sEVs were ultracentrifuged at 100,000×g for 60 min. Lastly, the supernatant was discarded, and PBS (200 µL) was added to resuspend the pellet.6) were seeded in 6-well plates. After 24 h, siRNA  and Lipofectamine RNAiMAXTM  diluted with OPti-MEM  were mixed in a 1:1 ratio. After 20 min at room temperature, siRNA and Lipofectamine RNAiMAXTM were added to the cell culture medium and incubated for 6 h. After 6 h, a culture medium without siRNA was added, and cell RNA and protein were extracted after 24 h. In addition, hUC-MSCs (1 × 105) were seeded on three-dimensional gelatin sponge scaffolds, and 10 scaffolds were co-cultured in 6 well plates. The siRNA transfection was performered according to the steps listed above, and the transfection was repeated every 3 days. After 14 days, the RNA, proteins, and sEVs were extracted.The hUC-MSCs  were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum (FBS). The cells were cultured at 37°C, and the medium was changed every 3 days when the cell confluence was approximately 80%.5 cells/well, and divided into 6 groups: Control group, LPS group, 2D-sEVs group, 4D-sEVs group, IGFBP2-siRNA-#2 and NSC74859 group. First, the LPS group, 2D-sEVs group, 4D-sEVs group, IGFBP2-siRNA-#2 and NSC74859 group were stimulated with 100 ng/mL lipo-polysaccharide  for 12 h, and the Control group contained an equal volume of medium without LPS. Next, the supernatant was removed and washed 3 times with PBS. STAT3 inhibitor, S3I-201 is used as described in the article (The RAW264.7 cells were seeded into 24-well plates at 1 × 10 article . NSC748511 (2D-sEVs) and (6.34 ± 0.63) x 1011 (4D-sEVs)  were provided by the Experimental Animal Center of Sun Yat-sen University. All animals received an intraperitoneal injection of 1% sodium pentobarbital (40 mg/kg) for anesthesia. After skin preparation, disinfection and fixation, a laminectomy was performed to expose the T9–T10 segments. A pressure device with a mass of 50 g was placed at the T10 segment for 10 min with a fixed spinous process. Then, we staunched the bleeding and sutured it layer-by-layer. 48 h later, a second operation was performed. After anesthesia, the pia mate was cut open, and the injured site of the spinal cord was gently flushed with saline to remove necrotic tissue. GelMA (20%) hydrogel  and 2.5% LAP  were mixed in a 1:1 ratio, and Dil dye-labeled 2D/4D sEVs (500 μg) were resuspended in the mixture. Injected with number of EVs corresponding to 500ug protein for each rat was (13.33 ± 3.50) x 104D-sEVs) . Next, tIn vitro, after removing the medium from the well plate, it was fixed with 4% PFA overnight and then washed three times with PBS. Immunohistochemistry was performed as described below: Briefly, the sections were rinsed with PBS, blocked with 10% normal goat serum, and 0.3% Triton X-100 in 0.01 M phosphate-buffered saline for 30 min at 37°C, and incubated with primary antibodies overnight at 4°C. Subsequently, the sections were rinsed with PBS and incubated with a secondary antibody for 1 h at 37°C. Finally, the sections were rinsed with PBS, coverslipped, and examined under a confocal microscope.Ten (10) days after the surgery , the animals were anesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. Then, spinal cords were removed, post-fixed, and dehydrated in sucrose/PB (30%). Longitudinal cryosections of the spinal cord, including the injury site, were cut at 20 μm and mounted on slides. The slides were dried and then soaked in Neutral Red staining solution  for 20 min, distilled water for 10 s, alcohol gradient decolorization for 5 s each 70%, 80%, 95%, and 100%, xylene transparent twice (each 4 min), and neutral resin sealing.For each group, the five transverse sections (5 rats/group) in the L1 segment of the spinal cord were selected by the method of one of ten. After neutral red staining, images were taken under light microscope (100x) and neurons of Clarke’s nuclei (CN) on 5 serial spinal cord sections of each rat were also counted (only neurons with nuclei were counted). There were five rats in each group.in vivo was performed by Laser Scanning Confocal Microscope  and ZEN 2.3 software (Carl Zeiss AG). One in every 10 longitudinal sections from each rat was processed (5 rats/group). After immunostaining with the respective markers, four randomized fields (200×) within the areas of the injury/graft site of the spinal cord were chosen for imaging and analysis. Immunofluorescence probing cells with identifiable nuclei (counterstained with nuclear dye Hoechst33342) were deemed immunopositive-stained cells. The percentage of CCR7, CD206, p-EGFR and p-STAT3-positive cells was calculated by dividing the number of double labeled positive cells  by the total number of CD68-positive cells. In vitro, the round coverslip with cells evenly distributed was divided into four quadrants, and a randomized field (200×) in each quadrant was selected for imaging and analysis. Triplicates were made for each study and the independent experiments were repeated for 4 times, and the positive percentage of cells was calculated by the same method in vivo.The quantitative analysis of CCR7, CD206, p-EGFR and p-STAT3-positive cell 7 cells) were lysed with pre-cooled (4°C) RIPA Buffer  supplemented with protease  and phosphatase inhibitor , centrifuged at 14000 g for 15 min (4°C), and the supernatant was transferred to a new centrifuge tube. The Protein A+ G Agarose  was diluted twice with PBS. Nex, add 100 μL Protein A + G Agarose beads to 1 mL total protein and shake slowly at 4°C for 2 h. After centrifugation at 14000 g for 15 min (4°C), the supernatant was transferred to a new centrifuge tube to remove Protein A + G Agarose. The extracted protein was diluted to 5 μg/µL in PBS, and 500 μL of each protein sample (5 μg/μL) was added to IGFBP2 (1:100), EGFR (1:100) and IgG (1:100), respectively. After incubation for 2 h at room temperature, Protein A + G Agarose was added and arose overnight at 4°C. The supernatant was discarded by centrifugation at 14000 g for 5 min (4°C), and the remaining precipitate was washed with 800ul PBS for 3 times. Lastly, it was resuspended with 60ul of loading buffer and boiled for WB detection.RAW264.7 cells  was added to lyse the cells. This solution was then boiled and stored in a −80°C refrigerator for later use. Western blot analysis was performed using the tissue samples for 10 days after surgery (4 rats/group). The spinal cords of 15 rats were immediately removed after deep anesthesia. The spinal cord segment (0.5 cm long) containing the injury/graft site was dissected and homogenized in lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 0.2% Triton X-100 and the Complete Mini Protease Inhibitor Cocktail. Equal amounts of proteins (20 μg) were separated by 10% SDS-PAGE and then transferred onto a PVDF membrane. After blocking non-specific binding sites with 5% nonfat milk in TBST (0.5% Tween 20 in TBS) for 1 h at room temperature, the membrane was incubated with primary antibodies overnight at 4°C, respectively. The primary antibodies were detected with an HRP-conjugated secondary antibody and then visualized with an enhanced chemiluminescence (ECL) western blot substrate kit .In vitro, the supernatant was collected from stimulated RAW264.7 cells. The inflammatory cytokines in the super-natant were measured using ELISA kits  according to the manufacturer’s instructions. The values of each protein detected in samples were normalized by the total protein value determined by the bicinchoninic acid method.After surgery, proteins were extracted from the spinal cord segments (0.5 cm) containing the injury site by ultrasonication (5 rats/group). The protein extract was ob-tained by centrifugation for 20 min at 12,000×g at 4°C. The extracted 2D sEVs and 4D sEVs (n = 3 for each group) were placed in a −80°C refrigerator. Proteomics analysis, including trypsin digestion, liquid chromatog-raphy-mass spectrometry (LC-MS), and data analysis, was performed by Jingjie PTM BioLabs.Protein Extraction: Sample was sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer . The remaining debris was removed by centrifugation at 12,000 g at 4 °C for 10 min. Fi-nally, the supernatant was collected and the protein concentration was determined with BCA kit according to the manufacturer’s instructions.Trypsin Digestion: For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM TEAB to urea concentration less than 2M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion.To perform detection and analysis, the tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column . The gradient was comprised of an increase from 8% to 22% solvent B (0.1% formic acid in 90% acetonitrile) over 20 min, 22%–35% in 13 min, and climbing to 80% in 4 min, then holding at 80% for the last 3 min, all at a constant flow rate of 600 nL/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific) coupled online to the UPLC. The elec-trospray voltage applied was 2.2 kV. The m/z scan range was 400–1,500 for the full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. The peptides were then selected for MS/MS using an NCE setting of 30, and the fragments were detected in the Orbitrap at a resolution of 17,500. The data-dependent procedure alternated between one MS scan followed by 20 MS/MS scans with a 30.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The fixed first mass was set as 100 m/z.p < 0.05 are significantly differen-tially expressed.The resulting MS/MS data were processed using the Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against the Homo_sapiens_9606_SP_20191115.fasta database concatenated with the reverse decoy and contaminants database. Trypsin/P was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set to 20 ppm in the First search and 4.5 ppm in the Main search, and the mass tolerance for fragment ions was set to 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification, and oxidation (M), deamidation , and acetylation  were specified as variable modifications. FDR was adjusted to <1%, and the minimum score for peptides was set to >40. Proteins with a fold change >1.5 and p < 0.05 means the difference is statistically significant.SPSS 13.0 was used for analysis, measurement data were presented as mean ± standard deviation (x ± s), and a one-way analysis of variance (One-Way ANOVA) was performed to compare between means. If the variances are homogeneous, the LSD method is applied for multiple comparisons of the means between groups; if the variances are not homogeneous, the Kruskal–Wallis test was applied to compare means among groups, and the Dunnett’s T3 method was for pairwise comparisons. p < 0.001, –10 (ug/particle) vs. (3.96 ± 1.2) x 10–10 (ug/particle), p < 0.001, The isolation and acquisition process for sEVs is shown in in vitro for in vivo tracking after injection. After injection into the spinal cord injury site for 7 days, most Dil-labeled sEVs were observed in the injury/injection site, with a small portion in the adjunct area that was rostral/caudal to the injury/injection site , Tuj1 (neurons), or Nestin   . Quantif+ cells. The immuno-histochemistry results showed that an increased portion of CD206+/CD68+ M2 macro-phages and a decreased portion of CCR7+/CD68+ M1 macrophages were observed in the injury/injection site of the spinal cord in the 4D-sEVs group relative to the SCI and 2D-sEVs groups  of the lumbar spinal cord segment 1 (L1) was selected for assessment because these large diameter neurons are morphologically readily identifiable, undergo degeneration approximately 1 month after a low thoracic lesion, and are sensitive to the treatments . Nissl sin vitro. RAW264.7 cells were activated by LPS and analyzed by adding sEVs from different groups. Red fluorescence was seen in the cytoplasm of RAW264.7 cells after adding DiI-labeled 4D sEVs after 3 h and lasted for at least 12 h  vs CD206+/CD68+ (8.36% ± 2.03%). However, after sEVs treatment, the M1 phenotype was suppressed despite LPS stimulation (INOS+/CD68+ (19.75% ± 8.39%) vs CD206+/CD68+ (18.27% ± 2.03%)) in the 2D-sEVs group and (INOS+/CD68+ (5.29% ± 2.02%) vs CD206+/CD68+ (41.12% ± 5.95%)) in the 4D-sEVs group. M2 polarization was more significant in the 4D-sEVs group compared with the 2D group (Lipopolysaccharide (LPS) effectively activates RAW264.7 cells towards the M1 phenotype. There was neither INOS (M1 phenotype) nor CD206 (M2 phenotype) expression in RAW264.7 cells without LPS stimulation (the Control group). The INOS expression was detected in RAW264.7 cells soon after LPS stimulation (the LPS group), showing an M1 phenotype polarization , LPS stimulation (the LPS group), 2D-sEVs, or 4D-sEVs with IGFPB2-siRNA pretreatment (the IGFBP2-siR-#2 group) . The exp+ M1 and CD206+ M2 macrophages were observed in both the IGFBP2-siR-#2 and 4D-sEVs groups  is critical for the M1 to M2 polarization of the activated macrophage. As a M2 dominantly expressed cytokine, IL-10 has been shown to inhibits nitric oxide formation by decreasing INOS protein expression in macrophage . The secs groups . However 4D-sEVs . ELISA r 4D-sEVs . These rin vivo is mediated by IGFBP2/EGFR protein signaling, we performed immunofluorescence assays. The results showed that IGFBP2/EGFR protein had been observed in the cells that engulfed sEVs (+ macrophages/activated microglia (To determine whether M2 microglial polarization at injury/injection sites fed sEVs , and p-Eicroglia . The fluicroglia . Similaricroglia . These rObtaining disease-targeting sEVs with promising efficacy remains a challenging task. Even though there has been positive feedback after their application in several diseases , dynamicThe 3D culture technique in our previous studies and the current one highlighted the “time” factor during the 3D culture process, a principle of “4D” biology . A homeoThe secondary injury cascades following the initial SCI result in a hostile microenvironment dominated by unrelieved neuroinflammation, which hinders the natural healing of the injured spinal cord. Studies have validated the anti-inflammation effect of the transplanted MSCs in the injured spinal cord . Among a4D culture technology generated more sEVs with larger sizes and varied proteo-mic profiles. The sEVs contained upregulated IGFBP2/EGFR proteins, which could po-larize macrophages/microglia from the pro-inflammatory M1 to pro-regenerative M2 phenotype via the EGFR/STAT3 pathway. This resulted in reduced neuroinflammation and neuronal loss at the spinal cord injury site. Therefore, the MSC 4D culture-derived sEVs technology could be developed into an effective treatment for SCI.


On the other hand, the inhibitors of the RSS-producing enzymes, such as 3-mercaptopyruvate sulfurtransferase and cystathionine γ-lyase, clearly enhanced ALIS formation and parthanatos. Interestingly, we found that Na2S4 activated heat shock factor 1 by promoting its dissociation from heat shock protein 90, leading to accelerated transcription of HSP70. Considering that the genetic deletion of HSP70 allowed the enhanced ALIS formation, these findings suggest that RSS prevent parthanatos by specifically suppressing ALIS formation through induction of HSP70. Taken together, our results demonstrate a novel mechanism by which RSS prevent cell death, as well as a novel physiological role of RSS in contributing to protein quality control through HSP70 induction, which may lead to better understanding of the bioactivity of RSS.Reactive sulfur species (RSS) have emerged as key regulators of protein quality control. However, the mechanisms by which RSS contribute to cellular processes are not fully understood. In this study, we identified a novel function of RSS in preventing parthanatos, a nonapoptotic form of cell death that is induced by poly (ADP-ribose) polymerase-1 and mediated by the aggresome-like induced structures (ALIS) composed of SQSTM1/p62. We found that sodium tetrasulfide (Na Parthanatos is a form of nonapoptotic programmed cell death governed by poly (ADP-ribose) polymerase-1 (PARP-1) . HoweverOxidative stress causes protein misfolding and then promotes the formation of the protein aggregates that are called aggresome-like induced structures (ALIS) . AlthougThe accumulation of reactive oxygen species (ROS) initiates oxidative stress that causes oxidation of cysteine residues in proteins. Recently, the critical roles of reactive sulfur species (RSS), such as cysteine hydropersulfide, that protect proteins from irreversible oxidation are highlighted . To date2Sn) can mimic endogenously produced polysulfides and thereby are used as RSS donors in experiments  are a group of highly conserved chaperone molecules that are induced in response to heat shock and other stresses . HSPs reIn this study, we found that RSS promote the induction of HSP70 expression at both protein and mRNA levels through the HSF1 activation and thereby suppress parthanatos by preventing the p62-based ALIS formation. Thus, our findings demonstrate a novel RSS-mediated inhibitory mechanism of cell death and a novel signaling mechanism that RSS activate HSF1 through the disruption of the HSP90–HSF1 complex.A and B, reduced cell viability upon CTX treatment was significantly restored by the PARP-1 inhibitor rucaparib or PARP-1 KO, showing that CTX induces parthanatos in HT1080 cells. Interestingly, we observed that cotreatment with Na2S4 restores viability of CTX-treated HT1080 cells in a concentration-dependent manner (C). Propidium iodide (PI) staining assays revealed that Na2S4 reduces PI-positive (dead) cells even at low concentration (1 μM) (D). Moreover, consistent with these observations, Na2S4 clearly inhibited nuclear translocation of AIF that triggers large-scale DNA fragmentation (E and F). Meanwhile, tumor necrosis factor-α in the presence of cycloheximide clearly promoted the cleavage of caspase-3 and PARP-1, while CTX failed to do so, further confirming that CTX induces parthanatos but not apoptosis (E). Collectively, these observations suggest that RSS produced by Na2S4 suppress CTX-induced parthanatos. We next examined the involvement of endogenously produced RSS in CTX-induced parthanatos. As shown in G and H, the loss of cell viability was enhanced, and the ratio of PI-positive cells was increased in the presence of I3MT-3 (HMPSNE), a potent and selective inhibitor of the RSS-producing enzyme 3-MST. In addition, I3MT-3 enhanced CTX-induced nuclear translocation of AIF (I and J). Similar to I3MT-3, the CSE inhibitor DL-propargylglycine (PAG) significantly enhanced the loss of cell viability induced by CTX and increased the ratio of PI-positive cells accompanied by enhanced nuclear translocation of AIF (K–N). Notably, inhibition of CSE strongly induced parthanatos even under conditions in which CTX alone failed to induce cell death (L). Therefore, endogenous RSS produced by CSE especially appear to contribute to the suppression of CTX-induced parthanatos.We have previously demonstrated that cefotaxime (CTX), a third-generation cephalosporin antibiotic, clearly promotes mitochondrial ROS generation and particularly induces oxidative stress-induced parthanatos in human fibrosarcoma HT1080 cell line , 33, 34.t manner C. Propidn (1 μM) D. Moreoventation , E and Fpoptosis E. Collecn of AIF , I and Jn of AIF , K–N. Noll death L. TherefA and B) (N-acetylcysteine or p62 KO as previously published data (A and C). On the other hand, rucaparib failed to inhibit the accumulation of p62 and K48-linked polyubiquitinated proteins in the detergent-insoluble fraction, whereas the accumulation of poly-ADP–ribosylated proteins was clearly inhibited (D).We next examined how RSS suppress CTX-induced parthanatos. Our previous study has demonstrated that oxidative stress-induced ALIS formation is required for CTX-induced parthanatos . As prevA and B) . The acched data , confirmhed data , A and Cnhibited D.Figure 2S4 also suppressed the accumulation of p62 and K48-linked polyubiquitinated proteins in the detergent-insoluble fraction (E). Moreover, immunofluorescent puncta of p62 and ubiquitinated proteins were clearly reduced by Na2S4 treatment (F and G). On the other hand, both I3MT-3 and PAG enhanced CTX-induced ALIS formation and led to increase the intensity of the costained p62 and K48-linked ubiquitin puncta (H–K). Thus, these results suggest that RSS suppress CTX-induced parthanatos by preventing the p62-based ALIS formation.Interestingly, Nafraction E. Moreovreatment , F and Gn puncta , H–K. ThA). However, interestingly, Na2S4 did not affect CTX-induced ROS generation . These results therefore suggest that additionally generated RSS by the Na2S4 treatment do not contribute to ROS elimination and prevent the p62-based ALIS formation by other mechanisms. We then speculated that RSS negatively regulate the p62-based ALIS formation at protein levels. In general, protein aggregates are removed by the autophagic or the ubiquitin-proteasome system. To test the possibility that RSS suppress the ALIS formation by activating these systems, the effects of Na2S4 on the ALIS formation were evaluated in the presence of the autophagy inhibitors bafilomycin A1 or the proteasome inhibitor MG132. As shown in Figure 3C, bafilomycin A1 elevated the expression levels of p62 in both detergent-soluble and detergent-insoluble fractions due to a lack of the lysosomal degradation of p62. Meanwhile, MG132 elevated the expression levels of p53 that is constitutively degraded by the proteasome . Interestingly, Na2S4 prevented the ALIS formation even in the presence of bafilomycin A1, whereas Na2S4 failed to do so in the presence of MG132, suggesting that the ubiquitin-proteasome but not the autophagic system is involved in the effects of Na2S4 (C and D). Accumulating evidence suggests that molecular chaperones mediate proteasomal degradation of misfolded proteins by delivering them to the 26S proteasomes (2S4 clearly elevates the expression of HSP70 protein in a time- and concentration-dependent fashion (E and F). Moreover, the upregulation of HSP70 by Na2S4 was more potent than that by CTX, and Na2S4 and CTX cotreatment additively upregulated HSP70 . Recent evidence has shown that PARP-1 is involved in the regulation of HSPs, including HSP70, which prompted us to test whether PARP-1 is required for the upregulation of HSP70 by Na2S4 . Moreover, K48-linked ubiquitin puncta were increased in HSP70 knockdown cells (K and L). These findings suggest that HSP70 inhibits the accumulation of the K48-linked polyubiquitinated proteins in the p62-based ALIS and further suggest the possibility that RSS disaggregates the ALIS by inducing HSP70.We therefore explored mechanisms by which RSS prevent the p62-based ALIS formation. As previous studies have demonstrated that RSS mitigate oxidative stress , both thFig. 3teasomes . We therteasomes , 37. Intby Na2S4 , 39. How2S4 strongly induces mRNA levels of HSP70 (A). Moreover, in addition to HSP70, Na2S4 induces HSP40 but not HSP90 at protein levels (B). These observations raise the possibility that Na2S4 acts on HSF1, a master regulator of HSPs. As expected, Na2S4 promoted nuclear translocation of HSF1, a transcriptional regulator of HSP70, which was not inhibited by PARP-1 KO (C and D). Interestingly, KRIBB11, recently identified as a HSF1 inhibitor (2S4-induced HSP70 upregulation at both protein and mRNA levels (E and F). Therefore, these results suggest that RSS upregulate HSP70 at mRNA levels through the HSF1-dependent mechanism. We next explored the mechanism by which RSS activate HSF1. Immunoprecipitation experiments revealed that Na2S4 promotes the dissociation of HSF1 from HSP90, which allows the nuclear translocation of HSF1 (G). The dissociation was confirmed by the coimmunoprecipitation assay of endogenous proteins (H). Moreover, the dissociation of HSF1 from HSP90 was observed by the in vitro treatment of Na2S4 (I). These observations raise the possibility that RSS directly act on the HSF1–HSP90 complex and execute their dissociation. Of note, recent reports have demonstrated that the chemical modifications of Cys 412, 521, and 564 in HSP90 allow the conformational changes of HSP90 protein  521 was substituted by alanine (A) failed to dissociate from HSF1 even in the presence of RSS. Moreover, the HSP70 induction induced by Na2S4 was strongly inhibited by the expression of HSP90 C521A mutant (K). Therefore, HSP90 C521A mutant appears to act as a dominant-negative mutant. Taken together, these findings further suggest that the persulfidation of HSP90 at Cys521 by RSS triggers the conformational changes of HSP90, leading to the dissociation of HSP1. Based on these results, we propose a model in which RSS prevent parthanatos mediated by the ALIS . These mechanistic findings suggest that RSS directly target and modify the cysteine residue of HSP90 to regulate the HSF1 activation. Accumulating evidence has shown that RSS alter the activity of proteins or GSH by persulfidation or polysulfidation of its cysteine residues . These observations suggest that RSS produced by 3-MST and CSE but not Na2S4 has an ability to eliminate ROS. Although further studies are required to explain this difference, distinct types of RSS may be produced by 3-MST, CSE, and Na2S4. 3-MST and CSE produce RSS via both hydrogen sulfide (H2S) and H2Sn, while Na2S4 basically produces RSS via only H2S4 , N-acetylcysteine, 2′,7′-dichlorodihydrofluorescein diacetate (WAKO), rucaparib, KRIBB11 (Selleck), I3MT-3 (MedChemExpress), PAG (Sigma-Aldrich). The antibodies used were against FLAG (Sigma), p62 (MBL), AIF, K48-linked ubiquitin, poly/mono-ADP Ribose , β-actin, PARP-1, Lamin A/C, HSP70, HSF1 (Santa cruz). Complementary DNAs (cDNAs) encoding human HSP90AB1 was obtained by performing PCR and was inserted into pcDNA3 with Flag tag plasmid as described previously , according to the manufacturer's protocol. The absorbance was read at 492 nm using a microplate reader. Data are normalized to control (100%) without stimulus, unless noted otherwise.Cell viability assay was performed as described previously . Cells w5/well. After indicated stimulation or treatment, cells were suspended in 1% Triton X-100 buffer  for 15 min. Cell lysates were centrifuged at 4 °C at 15,000 rpm for 15 min, and then the supernatants were collected as detergent-soluble fraction. After washing two times with 1% Triton X-100 buffer, the pellets were resuspended in Laemmli sample buffer  for 15 min. The lysates were centrifuged at 4 °C at 15,000 rpm for 5 min, and then the supernatants were collected as detergent-insoluble fraction. For preparation of whole cell lysates, cells were suspended in Laemmli sample buffer for 15 min, and then the lysates were centrifuged at 4 °C at 15,000 rpm for 5 min. The supernatants were collected as whole cell lysates. Protein concentration was determined using DC protein assay kit (Bio-Lab) according to the manufacturer’s instructions. Both the detergent-soluble and detergent-insoluble fractions and the whole cell lysate were subjected to immunoblot analysis as previously described (Cells were seeded on 12-well plates at a density of 2 × 10escribed .Immunoprecipitation was carried out with a modified procedure as previously described . After iHT1080 cells (1 × 10ˆ6 cells/one sample) were lysed in buffer A plus 0.2% Nonidet P-40 and 1 mm DTT as described above, and HSP90–HSF1 complex was immunoprecipitated using anti-HSP90 antibody with protein G-Sepharose beads. Beads were washed four times with PBS and then treated with indicated reagent. After reaction, beads were washed four times with PBS and subjected to immunoblotting with the indicated antibodies.5/well. For measurement of ROS generation, HT1080 cells were treated with indicated reagents and then incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (Wako) for 30 min. The cells were detached from the culture dish by trypsin treatment and dispersed by pipetting as mild as possible to avoid mechanical damages to the cells. Fluorescence intensity was measured by flow cytometry with the excitation wavelength at 488 nm and the emission wavelength at 580 nm. For PI staining, HT1080 cells were treated with CTX and Na2S4, I3MT-3, or PAG and then labeled with 2 μg/ml PI for 15 min. Fluorescent cells were detected by CytoFLEX (Beckman Coulter), and dead cells were analyzed by using CytExpert (Beckman Coulter).Fluorescence-activated cell sorting analysis was performed as described previously . Cells wKO cells were generated using the CRISPR/Cas9 system . Guide RFor determination of p62 mutation; forward: 5′-GAGGACTTTAGGGGGTCCCA-3′, reverse: 5′-AGGAATTAGCAGAGCGGCAG-3′. For determination of PARP-1 mutation; forward: 5′-GCATCAGCAATCTATCAG-3′, reverse: 5′-CTTCCCGGACACAGTTAA-3′.5/well. Cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 3% bovine serum albumin-PBS, and incubated with primary antibodies (anti-ubiquitin) overnight at 4 °C, followed by incubation with secondary antibodies  for 1 h at room temperature. The immunostained samples were enclosed with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI , and observed using a Zeiss LSM800 laser confocal microscope.HT1080 cells were seeded on 12-well plates at a density of 2 × 105/well. After indicated stimulation or treatment, cells were lysed in ice-cold lysis buffer containing 10 mM Hepes (pH 7.5), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, and 1% protease inhibitor cocktails  for 15 min. Cell lysates were added 1% NP-40 and then centrifuged at 4 °C at 2500 rpm for 3 min. After the supernatants containing cytoplasmic fraction were removed, the pellets were suspended in ice-cold lysis buffer containing 20 mM Hepes (pH 7.5), 400 mM NaCl, 1 mM EGTA, 1 mM DTT, and 1% protease inhibitor cocktails for 15 min vortexed every 5 min. Cell lysates were then centrifuged at 4 °C at 15,000 rpm for 15 min, and then the supernatants were collected as nuclear fraction.Nuclear extraction was performed as described previously . Cells w5/well. After indicated stimulation or treatment, cells were suspended in Sepasol-RNA I Super G  for 15 min, and Chloroform:isoamyl alcohol  was added. Thereafter, samples were vortexed and centrifuged at 4 °C at 15,000 rpm for 15 min. In this process, DNA is fractionated into hydrophobic fraction, and RNA is fractionated into hydrophilic fraction. RNA was precipitated by adding 2-propanol to the hydrophilic fraction, allowing to stand for 15 min, and then centrifuging at 4 °C at 15,000 rpm for 15 min. The RNA pellets were washed twice with 70% ethanol, air-dried, and dissolved in RNase free water. RNA purity was confirmed by Nanodrop ND-1000 (Thermo Fisher Scientific). Total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. Template cDNA was amplified by quantitative real-time PCR as described previously . All experiments were repeated at least three independent times. Two groups were compared using student’s t test. Multiple group comparisons were conducted using the one-way ANOVA followed by the Tukey–Kramer test using Prism software (GraphPad). Data were considered significant when ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.The value was expressed as the mean ± SEM using Prism software (GraphPad; The data presented in this study are available in article.The authors declare no conflict of interest.


Neutrophil granulocytes play key roles in innate immunity and shaping adaptive immune responses. They are attracted by chemokines to sites of infection and tissue damage, where they kill and phagocytose bacteria. The chemokine CXCL8  and its G-protein-coupled receptors CXCR1 and CXCR2 are crucial elements in this process, and also the development of many cancers. These GPCRs have therefore been the target of many drug development campaigns and structural studies. Here, we solve the structure of CXCR1 complexed with CXCL8 and cognate G-proteins using cryo-EM, showing the detailed interactions between the receptor, the chemokine and Gαi protein. Unlike the closely related CXCR2, CXCR1 strongly prefers to bind CXCL8 in its monomeric form. The model shows that steric clashes would form between dimeric CXCL8 and extracellular loop 2 (ECL2) of CXCR1. Consistently, transplanting ECL2 of CXCR2 onto CXCR1 abolishes the selectivity for the monomeric chemokine. Our model and functional analysis of various CXCR1 mutants will assist efforts in structure-based drug design targeting specific CXC chemokine receptor subtypes. Chemokines are small proteins secreted at sites of injury. Here, the authors describe the structure of the chemokine receptor CXCR1 bound to chemokine CXCL8, solved by cryo-EM. The model helps explain the ligand preferences of this receptor. More than 50 chemokines and their 23 receptors in humans are crucial to a very wide variety of physiological and disease processes, including development, immune function, inflammation, and cancer. Many chemokines interact with more than one receptor and vice versa, making a complex signaling network2. Chemokines share a common fold and at least four conserved cysteine residues, but are grouped into four subfamilies  based on the sequence motifs carrying signature cysteine residues in the N termini. CXC chemokines can be further subclassified as ELR+ or ELR− by the presence or absence of the tripeptide motif glutamic acid-leucine-arginine (ELR) N-terminal to the first cysteine. ELR+ CXC chemokines target neutrophils and are also known as neutrophil activating chemokines (NACs)3. Chemokine receptors are class A family GPCRs (G protein-coupled receptors) that recognise chemokines from different subfamilies. CXC chemokine receptors 1 and 2 (CXCR1 and CXCR2) were first described in 19915, and since then numerous studies have been conducted to investigate their therapeutic potential, for example in breast cancer6. CXCR1/2 are expressed by various immune and non-immune cells including neutrophils, macrophages, and endothelial cells. Both receptors bind the pro-inflammatory CXCL8, although CXCR2 interacts with a wider range of agonists.Chemokines are small proteins, 8–12 kDa in size, secreted by cells at sites of injury or infection to attract and activate leukocytes that carry receptors to these molecules+ CXC subfamily of chemokines, which dimerise under physiological conditions. It was one of the first chemokines to be discovered8. Formerly named neutrophil-activating factor (NAF)9, it was found to trigger a rapid and transient rise in the cytosolic level of free calcium in neutrophils  through a GTP-binding protein10. Evolutionary studies suggest CXCL8 is ancient, although functional diversification has led to it playing different roles in different species11. With a mature form only 72 amino acids in length and readily expressible in bacteria, it has been the subject of many structural studies by NMR and crystallography. The monomer folds into a three-stranded β-sheet with an α-helix at the C-terminus13. The N-terminus is disordered, and a region of coil from residues 10–22, called the N-loop, is found just upstream of the first β-strand. CXCL8 dimerises with Kd around 1–20 μM14, the dimeric form being held together by edge-to-edge contacts between β-strands to form a single 6-stranded sheet; the main tertiary structural difference from the monomeric form is a shift of the N-loop15. The single-site mutation R26C produces non-dissociating CXCL8 dimers held together by a disulphide bond between the two copies of residue 26, with a structure essentially identical to the native dimer16. CXCL8 incapable of dimerisation has been produced by various mutations, most simply by removing the last seven residues of the protein to disrupt the dimer interface17. Mutants unable to switch between oligomeric states are referred to as “trapped dimer” and “trapped monomer”. Previous work has revealed that monomeric CXCL8 interacts more strongly than the dimer with CXCR1 N-terminal peptide, and proposed that the dimerisation regulates binding to CXCR1 by reducing conformational flexibility of the chemokine18. Trapped CXCL8 monomer has been reported to bind CXCR1 ∼70-fold and CXCR2 ∼17-fold more tightly than a trapped dimer16. Nasser and colleagues found weaker selectivity, with the CXCL8 monomer binding CXCR1 approximately 6 times more tightly than the CXCL8 dimer, while CXCR2 showed a 4-fold preference for the CXCL8 monomer19. CXCL8 monomer was also found to elicit stronger responses, whether measured by phosphatidylinositol (PI) hydrolysis, secretion of β-hexasoaminidase or chemotaxis19. Monomers and dimers of CXCL8 induced similarly rapid internalisation and extent of phosphorylation of CXCR2, but the monomer proved more effective with CXCR119.CXCL8 is a member of the ELR20. In 2015, the first crystal structure of a chemokine receptor complexed with a chemokine was solved, of human CXCR4 bound to a viral chemokine antagonist, vMIP-II21. A number of X-ray crystallographic structures of chemokine receptors have since been described in ref. 24. Structural studies of the interactions between chemokines and their receptors have shown the receptor N terminus binds to the chemokine core at an interface called chemokine recognition site 1 (CRS1), while the chemokine N terminus sits within a pocket of the receptor TM helical domain . It has been shown by different groups that the receptor N-terminus plays an important role in chemokine specificity26. Both monomeric and dimeric forms of CXCL8 bind to CXCR2, and the structures have been solved of both complexes, with G proteins bound27. In the same paper, Liu and colleagues presented the crystal structure of CXCR2 bound to a small molecule allosteric antagonist, and with no ligand. Differences in the behaviour of CXCR1 and CXCR2 remain to be explained however, and we have therefore used cryo-EM to solve the structure of CXCR1 bound to CXCL8 and cognate G-proteins in order to clarify the functional contrast between these two closely related receptors.The structure of unliganded CXCR1 in phospholipid bilayers was solved by NMR in 2012R26C) under non-denaturing conditions, 7 + , 8 + , and 9+ ions of the dimer were observed, but no monomer ions  did not produce monomer peaks in the ESI-mass spectrum, indicating that the dimer is held together by disulphide bonds  were observed both for the native and acid-denatured samples  is exclusively dimeric, and trapped monomer (CXCL81–65) is exclusively monomeric, in solution.Various forms of CXCL8 were purified for structural and biochemical studies with CXCR1/2, and ESI-MS was conducted to determine the proportions of CXCL8 monomer and dimer. In the mass spectrum of the wild-type protein under non-denaturing conditions at a concentration of 2–7 μM, dimer peaks were clearly seen, together with less intense monomer peaks Fig. . The obsons Fig. . The obsnds Fig. . 5+ and les Fig. . The obs1–72 and Gαi1 protein, together with Gβ1 and Gγ2, stabilised by a single chain variable antibody fragment scFv16  end of TM5, and the N-terminal (cytoplasmic) end of TM6 . The second bond (C1103.25/C187ECL2) holds the N-terminal end of TM3 to ECL2 , but this is partially compensated by the introduction of L34N-term in CXCR2, which has no counterpart side-chain in CXCR1  or truncation of the N-terminus up to and including this residue increased the potency of the chemokine to inhibit adenylyl cyclase through both CXCR1 and CXCR2. Earlier work by Loos and colleagues also showed that citrullination of CXCL8 increases the ability of the chemokine to mobilise neutrophils30. R6CXCL8 sits between R2696.62 and R2807.28, but electrostatic repulsion will be reduced by D2656.58, whose side-chain is close enough to form hydrogen bonds with both R6CXCL8 and R2696.62.At CRS2, the CXCL8 N terminus interacts with the surface pocket formed between the TM helices of CXCR1 Fig. . S1CXCL8TM2 Fig. . Residue.58 Fig. . Unlike .58 Fig. . To confCXCL8 residue  requires about 85-fold and 32-fold higher concentration than the wild-type and the trapped monomer (CXCL81–65), respectively, to elicit a response from CXCR1 , it is seen that ECL2 of the receptor makes the only contacts with the partner CXCL8 subunit. This structured loop has a notably different sequence in CXCR1 and CXCR2 Fig. . CompariLFM, it iCR2 Fig. . This shing Fig. . Using tCR1 Fig. . When ECCR1 Fig. . Replaci31. Agonists trigger significant movements of TM5 and TM6 relative to the inactive state  within the conserved CWxP motif of TM6, close to the PIF motif and the NPxxY motif. The DRY motif in TM3 is also common to class A GPCRs, and helps regulate the switch between receptor states. Since CXCR1 and CXCR2 are highly similar, and have all the key motifs in common, the inactive state of CXCR2 can be used with confidence to model the behaviour of CXCR1  from the toggle tryptophan than is generally found with small molecule GPCR agonists  and trapped monomer (CXCL81–65) presented weakly charged ions with a narrow charge distribution, similar to those observed under non-denaturing conditions. The chemokine samples are therefore natively folded, and carry an intact N-terminal tail. This flexible region forms interactions with a surface pocket on the receptors, whose own flexible N-terminal tail binds to a surface groove on the chemokine. CXCL8 binding triggers calcium release and chemotaxis through both CXCR1 and CXCR233, but only CXCR1 mediates phospholipase D activation and respiratory burst34. These functional differences are mirrored by the preference of CXCR1 for the monomeric CXCL8, although no underlying connection is yet apparent. Although the native chemokine associates weakly, at higher concentration CXCL8 will compete with itself for binding to CXCR1, limiting its agonist effect, and it may be speculated that this allows gradient sensing.Chemokines are a very ancient family of proteins that may have arisen before jawed animals35. Granulocyte chemotactic protein 2 (GCP-2), better known as CXCL6, is a 75 residue protein with 31% sequence identity to CXCL8. It induces chemotaxis and calcium release in neutrophils through interactions with CXCR1 and CXCR2, which it binds with nanomolar affinity, but its role is less studied than CXCL837. CXCL6 triggers responses as effectively as CXCL8 does through CXCR2, but less so through CXCR1; replacing R20 of CXCL6  with glycine has no effect on binding to CXCR2, but renders the chemokine ineffective against CXCR137. Correlating such functional effects with the structural models is not trivial, and there are no obvious interactions made by K15CXCL8 of CXCL8 for example to suggest its importance. K11CXCL8 of CXCL8 on the other hand sits close to D26N-term of CXCR1. High-affinity binding alone however does not imply a highly effective agonist.The sequence differences between CXCR1 and CXCR2 are largely clustered into three segments: the N-terminal region, the second extracellular loop, ECL2, and the C-terminal cytoplasmic tail. Numerous studies have been undertaken to map the functional differences between the two receptors, and their interactions with chemokines, to these regions. GROα and NAP-2 show no effect with native CXCR1, but replacing the N-terminal peptide of the receptor with the sequence from CXCR2 allowed the chimeric receptor to respond to these chemokines as well as CXCL8In conclusion, CXCR1 and CXCR2 share common features of the activation mechanism found for other class A GPCRs. Our refined model reveals details of the interactions between CXCR1 and CXCL8, and shows that steric clashes with ELC2 are responsible for the known preference of the receptor for monomeric rather than dimeric chemokine. Although both CXCR1 and CXCR2 form similar agonist contacts in the CRS2 region, they have different N-terminal and ECL2 sequences and make substantially different interactions with CXCL8 at the CRS1 interface, which is largely responsible for chemokine selectivity.Spodoptera frugiperda (Sf9) cells, was synthesized by Genscript. The gene was cloned into a modified pFastBac HT-B vector containing N-terminal haemagglutinin (HA) signal sequence, Flag-tag (DYKDDDD), thermostabilized apocytochrome b562RIL (BRIL), HRC3V protease cleavage site and C-terminal 8 × His tag. CXCR1 was expressed in Sf9 insect cells using a Bac-to-bac expression system. Cells were infected at a density of 4 × 106 cells per ml and incubated at 27 °C for 60–72 h until harvest. To purify membrane fractions, the harvested cells were first disrupted in a hypotonic buffer containing 10 mM HEPES pH 7.5, 10 mM MgCl2, and 20 mM KCl, and then in a high osmotic buffer containing 10 mM HEPES pH 7.5, 1 M NaCl, 10 mM MgCl2, and 20 mM KCl in the presence of a protease inhibitor cocktail (Roche). Purified membranes were solubilized at 4 °C for 2 h in a buffer containing 50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 1% (w/v) n-dodecyl-beta-D-maltopyranoside , 0.2% (w/v) cholesterol hemisuccinate  with 2 μM CXCL81–72. Insoluble debris was removed by ultracentrifugation in 264,902 g at 4 °C for 1 h, and the supernatant was incubated with TALON® metal affinity resin (Clontech) overnight at 4°C. The resin was washed three times with 10 column volumes of washing buffer I containing 25 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 20 mM IMD, 0.05% DDM, 0.01% CHS, 2 μM CXCL81–72 and then washing buffer II containing 25 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 20 mM IMD, 0.05% LMNG, 0.005% CHS, 0.01% DDM, 0.002% CHS, 2 μM CXCL81–72 and then another 10 column volumes of washing buffer III containing 25 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 20 mM IMD, 0.05% LMNG, 0.005% CHS, 2 μM CXCL81–72. The protein was then eluted using the washing buffer III supplemented with 350 mM imidazole. Eluted protein was concentrated and passed through a PD-10 Desalting Column (Cytiva). HRV3C protease was added to cleave the Flag-tag and BRIL, and removed by nickel affinity chromatography. Fractions containing CXCR1 were pooled and subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated in 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 0.05% LMNG, 0.005% CHS in presence 2 μM CXCL81–72. The fractions containing CXCR1 were collected and concentrated before analysis.DNA encoding human CXCR1 (UniProtKB-P25024), codon-optimized for expression in E. coli BL21 (DE3) cells. The transformed cells were grown in LB medium at 37 °C. When the optical density at 600 nm reached 0.7, IPTG was added to 0.5 mM final concentration.DNA encoding human CXCL8 (UniProtKB-P10145) was synthesized by Genscript. The gene was inserted into the cloning site of pET-32a vector, such that the expressed protein carries an N-terminal sequence including a histidine tag followed by a TEV protease cleavage site (ENLYFQ | S). After TEV treatment, the expressed chemokine has the N-terminal sequence SAKELR. The plasmid was transformed into Harvested cells were then lysed by sonication in a buffer containing 20 mM HEPES pH 7.0, 150 mM NaCl, 0.2% Triton-X100, 0.25 μM PMSF. After centrifugation at 4 °C for 30 min, the supernatant was collected, and the CXCL8 was purified by Ni-NTA affinity chromatography. The eluate was treated with TEV protease, and the tag-free CXCL8 protein was applied to an anion exchange chromatography and Superdex75 increase 10/300 GL column (Cytiva), equilibrated with a buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl.38. The scFv16 gene was cloned into a modified pFastBac1 vector with N-terminal GP67 signal sequence and C-terminal TEV protease cleavage site followed by a 6 histidine-tag. Purification was carried out as before38 with simple modification. In brief, media containing secreted scFv16 was separated by centrifugation at 72-96 hours after infection. The pH of the medium was adjusted to 7.5-8.0 by adding Tris-base powder. Chelating agents were quenched by addition of 1 mM nickel chloride and 5 mM calcium chloride and stirring at room temperature for 1 h. After centrifugation, the supernatant was mixed and incubated with 5 ml of Ni-EXCEL resin (Cytiva). After 2 hours, the resin was collected and washed with 20 column volumes of buffer containing 20 mM HEPES pH 7.5, 500 mM NaCl and 20 mM imidazole. The scFv16 was eluted with 20 mM HEPES pH 7.5, 100 mM NaCl and 350 mM imidazole. After TEV protease treatment, the sample was further purified by size-exclusion chromatography using a Superdex 200 16/600 pg column (Cytiva). The peak fraction was collected and concentrated to 5 mg/mL for future use.The same construct was used for heterotrimeric Gαi1, Gβ1 and Gγ2 expression as a previous report1–72 and incubated at 25 °C for 30 min. Purified scFv16 was added to a 1:1.3, Gi heterotrimer:scFv16 molar ratio. The mixture was incubated on ice for 1 h. Then the mixture was subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 0.001% LMNG, 0.0001% CHS. Peak fractions containing CXCR1/Gi/CXCL81–72 complex were collected and concentrated to 2.5 mg/mL.CXCR1 and Gαi1 heterotrimer were mixed in a 1:1.2 molar ratio with 2.5 µl of Apyrase (NEB) in presence 2 μM CXCL8−1/px/sec. Magnification of micrographs was ×105,000, and pixel size was 0.83 Å/pixel. Defocus range was −0.8 to −1.6 µm. Data collection was automated using EPU software.3 µL of the sample was applied to a glow-discharged holey carbon grid . The grid was blotted for 5 s at 4 °C, 100% humidity and flash-frozen in liquid ethane using Vitrobot Mark IV instrument (Thermo Fisher Scientific) before storage in liquid nitrogen. Cryo-EM images were collected using a 300 kV Titan Krios G4 (Thermo Fishier Scientific) in Riken, Yokohama, Japan, equipped with a K3-summit camera with 15 eV slit. 4,175 movies were collected by standard mode for 48 frames with a total dose of 51.16 e/Å2, exposure time of 4.7 s, and dose on camera at 7.5 e39, beginning with Patch motion correction and CTF estimation. Micrographs under 5 Å CTF resolution were selected using Curate Exposures, giving 4,065 micrographs for analysis. Particles were auto-picked with blob picker, using all micrographs. 2,046,429 particles were extracted with binning state 3.35 Å/pix. Suitable particle classes from 2D classification were used to make 3D models ab initio. A 3D model of the entire protein complex was then used to re-run 2D classification. Using selected suitable 2D particle classes as reference, particles were auto-picked by Template picker from all micrographs. 2,530,947 particles were picked from micrographs with binning state 3.35 Å/pix. After further 2D classification, particles were selected and classified by Ab-Initio Reconstruction into 4 classes. Hetero-refinement was performed three times. One class showed the complete complex, and particles of this class were extracted with high-resolution (1.09 Å/pix). Subsequently Relion (v.4.0.0)40 was used for 2D classification of these particles, giving 282,687 in all. After multibody refinement focusing on the cytokine ligand, a new mask was made to cover the complete complex. 3D classification was performed with Relion, a class was chosen with strong ligand density. Finally, 120,631 particles were selected and 3D reconstruction performed with non-uniform refinement by cryoSPARC, and a 3.41 Å map was obtained. The final map for modelling was calculated by deepEMhancer (v.0.13)41.The collected data were processed by cryoSPARC (v.3.3.1)42 and Real-Space Refine in PHENIX43. The model was built manually based on Cα and side-chain maps using COOT44 and further refined using Real-Space Refine in the PHENIX suite.The model of CXCR1 was used from the AlphaFold Protein Structure Database (UniProt: P25024). Models of Gi heterotrimer and scFv16 complexes were taken from the structure of CXCR2/CXCL8(monomer) complex (PDB 6LFO). Models and cryo-EM map were roughly fitted by ChimeraXESI mass spectra were obtained with a SYNAPT G2 HDMS mass spectrometer equipped with a nanoelectrospray (nanoESI) ion source . Prior to nanoESI-MS, the protein solutions  were replaced with 200 mM ammonium acetate by gel filtration with Micro Bio-Spin 6  or dialysis. For obtaining the mass spectra under non-denaturing conditions, 5‒20 μM protein samples in 200 mM ammonium acetate were subjected to nanoESI-MS. For obtaining the mass spectra under denaturing conditions, the protein samples in 200 mM ammonium acetate were mixed with formic acid and methanol, resulting in 2‒3.5 μM proteins in 100 mM ammonium acetate containing 2‒3% formic acid and 30‒40% methanol. The pH of the protein solutions under the denaturing conditions was confirmed by pH test paper to be pH~1. A few microliters of the sample solution were deposited in a nanoESI emitter . The parameters used for the measurement were as follows: ion source temperature 70 °C; capillary voltage 0.7‒0.85 kV; sampling cone voltage 25‒40 V. Spectra were obtained by acquiring the data for 2 min in the mass range of m/z 500‒4000 (denaturing conditions) or 1000‒4000 (non-denaturing conditions). Mass spectra were processed using MassLynx 4.2 (Waters).45, in which the interaction between a Gα subunit and a Gβγ subunit was monitored by the NanoBiT system (Promega). Specifically, a NanoBiT-Gi1 protein consisting of Gαi1 subunit fused with a large fragment (LgBiT) at the α-helical domain (between the residues 91 and 92 of Gαi1) and an N-terminally small fragment (SmBiT)-fused Gγ2 subunit with a C68S mutation was expressed along with untagged Gβ1 subunit and CXCR1 or CXCR2 . HEK293A cells were seeded in a 6-cm culture dish at a concentration of 2 × 105 cells ml−1 (4 ml per well in DMEM (Nissui) supplemented with 10% fetal bovine serum (Gibco), glutamine, penicillin, and streptomycin), one day before transfection. Transfection solution was prepared by combining 10 µL (per dish hereafter) of polyethylenimine (PEI) Max solution , 400 µL of Opti-MEM (Thermo Fisher Scientific) and a plasmid mixture consisting of 400 ng CXCR1 or CXCR2 (or an empty plasmid for mock transfection), 200 ng LgBiT-containing Gαi1 subunit, 1 µg Gβ1 subunit and 1 µg SmBiT-fused Gγ2 subunit (C68S). For the lowered expression of the wild-type CXCR2, 40 ng plasmid (10-fold less) of CXCR2 plasmid was used. After incubation for 1 day, the transfected cells were harvested with 0.5 mM EDTA-containing Dulbecco’s PBS, centrifuged, and suspended in 2 ml of HBSS containing 0.01% bovine serum albumin  and 5 mM HEPES (pH 7.4) (assay buffer). The cell suspension was dispensed in a white 96-well plate at a volume of 80 µL per well and loaded with 20 µL of 50 µM coelenterazine (Angene) diluted in the assay buffer. After a 2 h incubation at room temperature, the plate was measured for baseline luminescence  and titrated concentrations of niacin  were manually added. The plate was immediately read at room temperature for the following 5 min, at measurement intervals of 20 s. The luminescence counts from 5 min to 10 min after ligand addition were averaged and normalized to the initial count. The fold-change values were further normalized to those of vesicle-treated samples and used to plot the G-protein dissociation response. Using Prism 9 (GraphPad Prism), the G-protein dissociation signals were fitted to a four-parameter sigmoidal concentration-response curve with a constraint of the HillSlope to absolute values less than 2. For each replicate experiment, Top and Bottom parameters of the wild-type, the trapped dimer and the trapped monomer of CXCL8 were constrained to be “shared values”. Thereafter, the parameters Span (= Top – Bottom) and pEC50  of individual receptor constructs were normalized to those of the wild-type CXCL8 performed in parallel and the resulting ∆pEC50 values were used to calculate ligand response activity of the ligands.CXCR1/2-induced Gi1 activation was measured by the NanoBiT-G-protein dissociation assay–1) and a goat anti-mouse IgG secondary antibody conjugated with Alexa Fluor 488 , as described previously in ref. 45. The resulting cells were analyzed by an EC800 flow cytometer equipped with a 488 nm laser (Sony). The fluorescent signals using all of the recorded events were analyzed with FlowJo software (FlowJo). Live cells were gated with a forward scatter (FS-Peak-Lin) cutoff at a value of 390 with a gain value of 1.7. Mean fluorescence intensity from all of the recorded events  was analyzed by a FlowJo software (FlowJo) and used for statistical analysis. For each experiment, we normalized an MFI value of the mutants by that of the wild-type performed in parallel and denoted relative levels.Transfection was performed according to the same procedure as described in the ‘NanoBiT-G-protein dissociation assay’ section. After incubation for 1 day, the transfected cells were detached and then dispensed in a 96-well V-bottom plate and fluorescently labeled by using an anti-FLAG epitope (DYKDDDDK) tag monoclonal antibody (Clone 1E6, FujiFilm Wako Pure Chemicals; 10 μg mlFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Bacillus subtilis, Microbial Type Culture Collection and Gene Bank (MTCC 1305) and Gram-negative Escherichia coli (MTCC 443). Synthesis was followed by aza-Michael addition reaction, where the acrylonitrile accepts an electron pair from the respective amines and results in the formation of n-alkyliminobis-propionitrile and n-alkyliminopropionitrile under microwave irradiation. Characterization of the compounds were performed using Fourier Transform Infrared (FTIR), Proton Nuclear Magnetic Resonance (1H NMR) and Electrospray Ionisation Mass Spectrometry (ESI–MS). The particle size characterization was done by Dynamic Light Scattering (DLS) technique. The antibacterial study showed higher inhibition rate for both Gram-positive and Gram-negative bacteria. The antibacterial ability was found to be dose dependent. The minimum inhibitory concentration against both bacteria were found to be 1, 3, 0.4, 1, 3 µl/ml for E. coli and 6, 6, 0.9, 0.5, 5 µl/ml for B. subtilis. Time-kill kinetics evaluation showed that the adducts possess bacteriostatic action. Further it was evaluated for high-throughput in vitro assays to determine the compatibility of the adducts for drug delivery. The haemolytic and thrombolytic activity was analysed against normal mouse erythrocytes. The haemolytic activity showed prominent results, and thereby projecting this acrylonitrile adducts as potent antimicrobial and haemolytic agent.In this work, five acrylonitrile adducts were screened for antibacterial activity against Gram-positive  If current trends continue, bacterial infections with multidrug-resistant traits will be listed among the top three global public health risks, causing up to 10 million fatalities worldwide by 20505. Antimicrobial resistance against accessible medications has evolved from the availability of poor treatment and widespread usage of antimicrobial agents. There are various reports on studies of nanocomposites and nanoparticles as effective antimicrobial agent. Hasanin et al. has reported several works on doping of nanocomposites and showed that doping a material with the nanocomposite enhances the antimicrobial activity against various gram positive and gram negative bacteria6. They have proved that silver nanoparticles (Ag-NPs) loaded nanocomposites has high potential as antimicrobial agent7. Loading of the nanocomposites was carried out through ecofriendly method9 and incorporation of the nanocomposites enhances the antimicrobial, antifungal, antiviral, antioxidant and anticancer activities11. Samuel et al.12 developed simple one-pot method and promoted the biological synthesis of nanoparticles as a promising method for nanoparticle synthesis. They have developed a hybrid polymer composite of lithocholic acid (LCA), zinc pyrithione (Zn) and cinnamaldehyde (Cn) (named as LCA–Zn–Cn) which displays potent antibacterial/antibiofilm activity13. Moreover, they have biosynthesized iron oxide nanoparticle (Fe3O4 NPs) and showed its dual benefits as effective adsorbent for removal of heavy metal ion from wastewater and as biomedical agents12.In recent years, the majority of infectious diseases have been linked to bacteria, as a result of widespread antibiotic resistance among bacteria strains. According to the 2016 United Nations (UN) declaration on antimicrobial resistance14. The adduct 2-(3-hydroxy-2-oxoindolin-3-yl)acrylonitrile (ISACN) has shown as a promising anti-inflammatory agent15. MBHA obtained using acrylonitrile as Michael acceptor inhibited the growth of various species of bacteria and fungi showing minimum inhibitory concentration of 32 µg/ml which reflects the antimicrobial effectiveness of acrylonitrile adducts14. The acyl derivatives enantiomers of the Morita–Baylis–Hillman adduct (±)-2-[Hydroxy(m-nitrophenyl)methyl]acrylonitrile and (±)-2-[Hydroxy(p-nitrophenyl)methyl]acrylonitrile showed no toxicity in peritoneal macrophages from Swiss mice16. Rocha et al.17 has reported that MBHA exhibits no haemolytic activity to human erythrocytes. These adducts can serve as a potential drug candidate for treating various diseases.Morita–Baylis–Hillman adducts (MBHA) derived from isatin derivatives and acrylonitrile has showed bactericidal and fungicidal properties by preventing the growth of the majority of microorganism species utilized in biological experiments18. The major-league in the structural diversity of N-heterocyclic compounds made it recognizable as an effective molecule in the search of new drugs19. Large numbers of clinically approved drugs have developed multidrug resistance leading to adverse side effects. In medicinal chemistry, investigation on nitrogen-based molecules has progressively become an attractive area of research. Study on the antibacterial activity of the alkaloids shows the efficacy of the nitrogen constituent in the molecule20. Metronidazole and quinolones are prominent antibacterial drugs derived from naturally occurring alkaloids21. Thus, many pharmaceutical significant drug molecules contain ‘Nitrogen’ as a constituent in the molecule. Bioactive nitrogen containing components shows potent antimicrobial activity against gram-positive and gram-negative bacteria24. Advantage of having a nitrogen atom and proton-donating amine hydrogen atoms in the molecule is that it facilitates the formation of hydrogen bonds with enzymes, proteins and receptors21. On a similar note organic molecules containing –C≡N as the functional group, such as nitriles, constitutes a series of diverse organic molecules with diverse structures29. The medical world uses a variety of nitrile-containing drugs with various structures32. Medicinal compounds with unsaturated –C≡N can be conjugated either with additional electron withdrawing groups or heteroatoms. Egelkamp et al.33 reported effects of various nitrile containing compounds on bacterial communities. They made a comparative study between the nitrile compounds and their corresponding degradation products. The study revealed distinct impact of nitrile groups on the bacterial communities compared to the compounds with carboxylic acid groups. Therefore, there is sufficient scope to investigate the efficacy of nitrile compounds as antibacterial agent34.Investigation of aza-Michael adducts in the field of biological studies has not been explored much. Thus the noticeable lack of novel compounds as effective antimicrobials to suppress the microbial growth encouraged us to evaluate the acrylonitrile adducts as efficient antimicrobial agents. The use of nitrogen based molecules in the field of pharmaceuticals is expanding day-by-day35. We have developed a series of aza-Michael adduct which has potent antimicrobial activity and shows no haemolysis. One-pot synthesis, short reaction time, cost-effectiveness, easy availability, antimicrobial and biological assay of acrylonitrile adducts marks the novelty of the work. These adducts have multiple applications. It has been reported that the adducts showed prominent adsorption of Fe2+ ion from aqueous media36. In addition to it, this work aims to investigate the activity of acrylonitrile adducts against antimicrobial and biological properties. Till date, very few reports are available on the application of acrylonitrile adducts as potent antimicrobial agent which prompted us to undertake this study with a series of aza-Michael adducts to evaluate the efficiency of such molecules specifically for bio-medical applications. The aza-Michael adducts used for the study were synthesized under microwave irradiation using literature procedure35 under mild conditions. Furthermore, the five acrylonitrile adducts were also investigated for biological studies. The breakdown of red blood cells caused by the rupture of the lipid bilayer of the cell membrane and releasing of intracellular contents is called haemolysis. Because of easy availability, isolation and having similar properties to those of human cells, the in vitro analysis with the erythrocyte of mouse blood is a beneficial tool38 for assessing the efficacy of the synthesized compounds for preclinical trials39. This study directly indicates the toxicity of injectable drugs. Haemolytic activity was evaluated for acrylonitrile adducts and interaction of the compounds with erythrocyte of mouse blood cell was recorded. We have also carried out the investigation on thrombolytic behaviour41. As the compounds synthesized from aza-Michael addition reaction are proposed to be used as intermediates for drugs having properties like anti-inflammatory, antibiotics, antimicrobial etc., therefore the blood clot dissolving activity was evaluated for all the synthesized acrylonitrile adducts42.Aza-Michael addition reaction can be considered as a simple as well as convenient synthetic route for synthesis of novel compounds through C–N bond formation. The reaction between several primary aliphatic amines and acrylonitrile has been carried out in our previous work254 plates were used for thin layer chromatography. All the chemical reagents were of analytical grade and were used without further purification. Solvents used during the synthesis were dried by using standard procedure. Mixing of the substrates was carried out by Light-Emitting Diode (LED) Digital Vortex Mixer.All the chemicals used for the synthesis of mono and di-adducts of acrylonitrile were procured from Sigma-Aldrich and Tokyo Chemical Industry (TCI) Chemicals, Japan. Molecular sieves of 4 Å beads, 4–8 mesh were purchased from Sigma-Aldrich. Merck silica gel 60 FBacillus subtilis (MTCC 1305) and Gram-negative bacterial strain Escherichia coli (MTCC 443). Both of the strains were procured from Microbial Type Culture and Collection (MTCC), Chandigarh, India. Maintaining a temperature of 37 °C lyophilized culture was revived in nutrient broth medium by keeping it in a rotatory shaker for 12 h. Using 0.5 McFarland standards each strain was adjusted to a concentration of 108 cells/ml45.The compounds were evaluated for their antimicrobial activities against two bacteria viz. Gram-positive bacterial strain 8 cells/ml. 50 µl of each bacterial culture was uniformly spread on the Nutrient agar (NA) plates after solidification. Once the bacterial culture on each plate was settled down, 100 µl of 1 g/10 ml ceftriaxone (Positive control) and 100 µl of water (negative control) was loaded in the well as a reference. 100 µl of different concentration  of acrylonitrile adducts were loaded on bacterial plates and then these plates were incubated at 37 °C for 12 h. The diameters of zone of inhibition in each plate were recorded in millimetre (mm)44.The fundamental evaluation for antimicrobial activity of all the compounds was done by agar well diffusion assay. Bacterial cell cultures were inoculated in nutrient broth and kept for approximately 24 h in incubator shaker to attain the concentration of 108) cells/ml was pellet down at 8000 rpm for 15 min. The supernatant layer was discarded, and the remaining broth was removed by washing the pellet thrice. To make up the stock volume, the pellet of each bacteria was suspended with 5 ml of distilled water. Additionally, 10 µl of the bacterial culture was added in to 2 ml of acrylonitrile adducts solution of different concentrations  in 15 ml test tube. The parameters of temperature 37 °C, time 24 h at 120 rpm was taken for incubation of the test tubes in shaker incubator. The presence of viable bacterial cells was measured using plate count method47. The effect of concentration of acrylonitrile adducts on bacteria removal efficiency was calculated by percentage reduction as follows:5 ml of the stock culture The synthesized acrylonitrile adducts were analysed for haemolytic activity against normal mouse erythrocytes as per International Organization for Standardisation (ISO) 10993-4 standards. In the context of this research, the mixture of two compounds, mouse blood (3 ml) and 1× Phosphate-Buffered Saline (PBS) (5 ml) was centrifuged at 3000 rpm for 5 min. The pellets were suspended in 5 ml of PBS, and then the sample solution (20 µl) in dimethyl sulphoxide (DMSO) was added to 180 µl of this combination. The mixture was incubated for 30 min at 37 °C, then centrifuged for 5 min at 13,000 rpm and refrigerated. A spectrophotometer was used to detect the free haemoglobin in the supernatant at 576 nm. The negative and positive controls were DMSO and 1% Triton X-100, respectively. The percentage haemolytic activity was calculated using Eq. . A 100 µl of test solution in 10 ml of DMSO was added to the clot, which was then incubated at 37 °C for 180 min. The liquid portion of the tube was discarded after incubation, and the weight of the tube (z) was measured again to quantify the difference in weight before and after thrombolysis, which was expressed as %thrombolysis. DMSO and streptokinase (SK), were taken as the negative and positive control respectively. The % Thromobolysis was calculated as per Eq.  as internal standard and the δ values are reported in ppm. Mass spectral data were recorded on Waters Ultra-Performed Liquid Chromatography-Triple Quadrupole Mass Spectrometry (UPLC-TQD) (ESI–MS) mass spectrometer.The synthesized acrylonitrile adducts were characterized by FTIR, 35 as shown in Fig. n-Alkyliminobis-propionitrile and n-alkyliminopropionitrile was synthesized by aza-Michael addition reaction under microwave irradiation using literature procedure−1 can be attributed to stretching due to –C≡N bond based on available literature51. The conjugate addition of nitrogen nucleophile, n-propylamine with the Michael acceptor i.e. acrylonitrile leads to the formation of n-alkyliminobis-propionitrile and n-alkyliminopropionitrile. Thiyagarajan et al. employed several nucleophiles with acrylonitrile to yield the corresponding Michael adducts52. The successful occurrence of aza-Michael addition reaction of acrylonitrile with n-alkylamines was confirmed by the presence of the –C≡N stretching at 2247 and 2251 cm−1, which resulted in the formation of alkyliminobis-propionitrile and n-alkyliminopropionitrile. Nitrile bearing compounds are known to possess better inhibitory power against gram positive and gram negative bacteria. The –C≡N stretch for alkyliminobis-propionitrile and n-alkyliminopropionitrile are shown in the Fig. FTIR spectra of n-propyliminobis-propionitrile (N1), n-butyliminobis-propionitrile N2), n-hexyliminopropionitrile (N3), n-decyliminopropionitrile (N4) and n-dodecyliminopropionitrile (N5) are shown in Fig. , n-hexyl1H NMR characterization highlights the presence of different functionality in the resulting adducts. Guo et al. carried out Michael addition reaction between diethylamine and acrylic acid 2-phenylsulfanyl-ethyl ester to form 3-Diethylamino-propionic acid 2-phenylsulfanyl-ethyl ester53. From the spectroscopic data they revealed that the 1H NMR signal for the alkyl group protons attached to the α position of tertiary nitrogen atom appears in the range δ = 3.12–3.74 ppm. On evaluating the 1H NMR spectra for five acrylonitrile adducts, it was observed that 1H NMR shift for the CH2 group attached to α position of tertiary nitrogen appeared at chemical shift value ranging from δ = 2.46–2.9 ppm and the same for the CH2 group attached to β position appeared at δ = 1.44 ppm. Results of analysis from 1H NMR spectra of n-alkyliminopropionitrile also confirm the presence of α-CH2 groups attached to the nitrile group, from similar reasoning. 1H NMR peak for this group was observed in the range δ = 2.73–2.92 ppm and the corresponding peak for β-CH2 group was observed at δ = 2.99 ppm. 1H NMR peak for the terminal methyl protons attached to alkyl chain was observed in the range δ = 0.88–0.90 ppm. Proton NMR spectra for all the five acrylonitrile adducts (N1 to N5) were found identical with the ones reported in literature35  studies provided further convincing evidence for the synthesis of all the five acrylonitrile adducts. e35 Fig.  and ther2]+ Fig. . The parE. coli and B. subtilis are shown in Figs. E. coli (18 ± 0.1) and B. subtilis (17 ± 0.3 mm)  Fig. .Figure 4E. coli, the diameter of the zone of inhibition for N1 at 2%, 4%, 6% and 8% are 10 ± 0.2 mm, 11 ± 0.1 mm, 12 ± 0.2 mm and 13 ± 0.3 mm respectively as shown in Fig. In case of B. subtilis, the diameter of the zone of inhibition for N1 at 2%, 4%, 6% and 8% are 6 ± 0.3 mm, 7 ± 0.1 mm, 8 ± 0.1 mm and 9 ± 0.2 mm respectively as shown in Fig. Similarly, in case of The result of this study strongly reveals the antibacterial activity of acrylonitrile adducts at low, medium and high dose as shown in Tables Bacillus megaterium and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Proteus vulgaris)55. In addition, the antifungal activity (Aspergillus niger) was also checked. They reported that for the gram-positive bacteria the diameter of the zone of inhibition was observed to be in the range of 10 to 21 mm while for the gram-negative bacteria the same was obtained in the range of 10 to 22 mm. In case of A. niger55 the zone of inhibition was observed to be in the range of 10 to 18 mm. In another study, Ammar et al.56 checked the antimicrobial activity of sulfonamide containing pyrazolopyrimidine, pyrimidine and pyrazolotriazine derivatives. Only two compounds showed good antibacterial activity (zone over 3.0 mm) against Serratia marescens and Proteus mirabilis while the rest of the compounds were showing antibacterial activity in the range of 0.6 to 1.4 mm56.Similar to this study, novel nitriles and imidazolines were prepared and their antimicrobial activity was checked against Gram-positive , n-butyliminobis-propionitrile (N2), n-hexyliminopropionitrile (N3), n-decyliminopropionitrile (N4) and n-dodecyliminopropionitrile (N5) removed the E. coli with reduction of 10, 14.22, 10, 7.3 and 10.1 percentage respectively at 0.5 h. On further increasing the incubation time the percentage reduction at 24 h against E. coli for N1, N2, N3, N4 and N5 was recorded as 87.3%, 89.7%, 89%, 87.6% and 95% respectively. On increasing the amount to 4%, antibacterial activity increased from 19 to 89.9%, 16.5 to 90.7%, 26 to 99.9%, 20.7 to 90.5% and 18 to 96% for N1, N2, N3, N4 and N5, from 0.5 to 24 h respectively. On further increasing the amount to 6%, the percentage reduction was observed as 22 to 96.2%, 20 to 99%, 53 to 99.999%, 28.1 to 99%, 28 to 99.1% for N1, N2, N3, N4 and N5 from 0.5 to 24 h respectively. When the concentration of acrylonitrile adducts was further increased to 8%, the percentage reduction was found to be 100, 99.99, 100, 99.99 and 100% against E. coli after incubation time of 24 h with 100 ml of media.In case of B. subtilis, the 2% of N1, N2, N3, N4 and N5 removed B. subtilis with reduction of 9.6, 7.1, 10, 10.8 and 18.8 percentage respectively at 0.5 h. Increasing the incubation time further to 24 h, the percentage reduction against B. subtilis was increased to 80.11%. On increasing the amount of N1, N2, N3, N4 and N5 to 4%, antibacterial activity increases from 16.5 to 89.5%, 14.3 to 85.4%, 15 to 90.9%, 19.9 to 90.5% and 19.5 to 90%, from 0.5 to 24 h respectively. On further increasing the amount to 6%, the percentage reduction was found to increase from 23.9 to 99.6%, 20.4 to 90.3%, 27 to 99.99%, 35.7 to 99.99% and 20.5 to 99% for N1, N2, N3, N4 and N5, from 0.5 to 24 h respectively. Finally, further increase in concentration of N1, N2, N3, N4 and N5 to 8%, significant change of percentage reduction was noted which were recorded as 99.9, 99.9, 100, 100 and 99.99 percentage reduction against B. subtilis, after incubation for 24 h with 100 ml of media.In case of The results of antibacterial activity of both bacteria under dynamic condition reveals that all the bacterial cells were removed by N1, N2, N3, N4 and N5 after contacting for 24 h.10 cfu/ml) values for E. coli and B. subtilis. It is mathematical term used to express the relative count of living microbes eliminated by antibiotic. For instance, if the number of cfus in the control sample was found to be 106 and after using antibiotic the test sample was having 105, that would be a Log reduction of 1 or a reduction of 90%. In the present study, the bactericidal activity of acrylonitrile adducts were evaluated by the time-kill kinetic analysis. The reduction in bactericidal activity was evaluated by determination of the survival rate [log10 cfu/ml vs Time(h)] of bacteria when exposed to a drug.Figures E. coli respectively and for B. subtilis it was reduced by 0.04, 0.03, 0.05, 0.05 and 0.09, at 0.5 h. While at 4% v/v N1, N2, N3, N4 and N5 treatment, the considerable growth inhibitory response was observed at 24 h in both Gram positive (1 log10 cfu/ml) and Gram negative (3 log10cfu/ml) bacteria. On further increasing the concentration of N1, N2, N3, N4 and N5 to 6% v/v, log reduction observed for E. coli was 1.42, 2, 5, 2 and 2.05 log cfu/ml respectively and the corresponding values for B. subtilis was noted as 2.4, 1.01, 4, 4 and 2 log cfu/ml respectively. Increasing the concentration further to 8% (V/V), log reduction values were dramatically improved to 5, 4, 8, 4 and 5.4 log cfu/ml in case of E. coli while in case of B. subtilis the values were obtained as 3, 3, 6, 6 and 4 log cfu/ml reduction. Based on the growth pattern, the action of 2%, 4%, 6% and 8% v/v on N1, N2, N3, N4 and N5 indicates a bactericidal effect.At 2% v/v N1, N2, N3, N4 and N5 treatment, the cell population was reduced by 0.05, 0.07, 0.05, 0.03 and 0.05 for 61, 5–10% low haemolysis and > 10% marked haemolysis62. In vitro biocompatibility assessment of acrylonitrile adducts were carried out by direct contact with erythrocytes using PBS and the %haemolysis is shown in Fig. In the present study five acrylonitrile adducts such as n-propyliminobis-propionitrile (N1), n-butyliminobis-propionitrile (N2), n-hexyliminopropionitrile (N3), n-decyliminopropionitrile (N4) and n-dodecyliminopropionitrile (N5) were used to check its haemolytic activity with normal erythrocytes of mouse. Haemolysis is a process occurring as a result of contact of blood with a foreign object and as per the standards of American Society for Testing and Materials (ASTM) if the percentage of haemolysis is < 5% then it is considered as no haemolysisThe positive control and negative control in this study was 1% TritonX-100 and 1× PBS respectively and their absorbance is mentioned in Table 63 synthesized pentasubstituted pyrroles and subjected to in vitro anti-thrombotic analysis. The divergent pyrroles 2-(2-argio-1-methyl-5-(methylthio)-4-nitro1H-pyrrol-3-yl)acetonitrile derivatives exhibited excellent clot lysis. Danac et al.64 has synthesized cyano-substituted pyrrole fused (iso)quinolone derivatives. Anticancer study showed that fumaronitrile has potent anticancer properties. It exhibited antiproliferative action against cancer cell lines. The thrombolytic activity was evaluated and it was found that the blood dissolving activity was less than the negative control so the adducts did not showed any thrombolytic activity as shown in Table Shanmugam et al.Successful synthesis of five acrylonitrile adducts have been demonstrated in this work, using environmentally benign synthetic method. The novelty of the compounds was exhibited in terms of their excellent antimicrobial activity against both the bacterial stains. Most importantly, during the study, the reported compounds were also established as potent haemolytic agent, which significantly enhances the pharmaceutical and biological significance of the compounds. All these compounds showed least percentage of haemolysis, 0.06 to 2.97%, which is safe for clinical trials. This fact other way indicates that the acrylonitrile adducts successfully can be employed for drug delivery. Study on the bactericidal characteristics of the compounds indicated that for all the five acrylonitrile adducts, complete removal of both the gram-positive and gram-negative bacteria were possible with an 8% concentration of the compounds, after 24 h, which strongly reveals the bactericidal characteristics of the compounds. Summing up, we have developed a series of acrylonitrile adducts which can be explored as efficient antimicrobial and haemolytic agent.Supplementary Information.