The APC-related markers HLA-DR, CD27, CD80, and CCR7 on fresh γδT cells were significantly higher in patients with sepsis compared with matched controls; however, they responded poorly to 4-hydroxy-3-methyl-2-butenyl pyrophosphate (HMBPP) stimulation, characterized by the deactivation of these APC markers and impaired proliferation. Furthermore, the adhesion function of γδ T cells, essential for antigen presentation, was greatly reduced in patients with sepsis; for instance, in co-cultures with green fluorescent protein-expressing Escherichia coli, HMBPP-activated γδT cells from healthy individuals adhered to E. coli efficiently, whereas no such phenomenon was observed with respect to γδT cells from patients with sepsis. In line with these results, in co-cultures with isolated CD4+ αβ T cells, HMBPP-activated γδT cells of healthy individuals promoted the efficient proliferation of CD4+ αβ T cells, whereas γδT cells from patients with sepsis did not do so. In conclusion, our findings show that the antigen-presenting function of γδT cells is severely impaired in patients with sepsis and the mechanisms behind need further study.Impairment of antigen-presenting functions is a key mechanism contributing to sepsis-induced immunosuppression. Recently, γδ T cells have been demonstrated as professional antigen-presenting cells (APCs); however, their role in sepsis remains unknown. In this  E. coliefficiently and promoted the efficient proliferation of CD4+ aß T cells, whereas γδT cells from patients with sepsis did not do so. In conclusion, our findings show that the antigen-presenting function of γδT cells is severely impaired in patients with sepsis.The APC-related markers on fresh γδT cells were significantly higher in patients with sepsis compared with matched controls; however, they responded poorly to 4-hydroxy-3-methyl-2-butenyl pyrophosphate (HMBPP) stimulation, characterized by the deactivation of these APC markers and impaired proliferation. HMBPP-activated γδT cells from healthy individuals adhered to   The latest definition of sepsis highlights the role of dysregulated host immune responses to infection in the context of organ dysfunction aggravation . Sepsis The antigen-presenting process is the bridge connecting innate and adaptive immunity. Invading pathogens can be recognized and presented to T cells by professional antigen-presenting cells (APCs), such as dendritic cells (DCs). This process rapidly activates the adaptive immune responses, indispensable for the elimination of invading pathogens. Of note, impairment of the antigen-presenting function has been known to play important roles in the pathogenesis of sepsis . In factin vitro studies have reported that activated γδ T cells possess unique and powerful antigen-presenting features, including the high expression of different APC markers , and promote the proliferation and activation of CD4+ and CD8+ T cells ). Categorical variables are presented as the frequency. Comparisons of the proportion of γδ T cells and the expression of surface markers between healthy individuals and the ones with sepsis were carried out using ANOVA. Additionally, the paired-sample mean test was used to evaluate the differences in the number of cells and the expression of surface markers in the context of individuals with sepsis and the control group, before and after stimulation. Data were analysed using the GraphPad Prism  or SPSS  software. Forty-two patients with sepsis and 27 healthy volunteers were enrolled in this study. The age and sex distribution were similar between patients with sepsis and healthy controls: 46 (28–57) versus 37 (26–43) years and 64.29% versus 59.26% males, respectively. Overall, patients with sepsis were severely ill with APACH II and SOFA scores of 18.13 (± 5.27), and 8.38 (± 1.15), respectively. Of note, patients with septic shock accounted for 52.38% of the total patients. Additionally, the abdomen was the most common infection site (50% of cases). A bacteriological confirmation was obtained in 92.86% of patients with sepsis and Gram-negative bacterial infection accounted for 54.76% of them. The time between acute organ dysfunction to inclusion was 5 (2–7) days. The patient demographics and clinical characteristics are listed in P < 0.01; CD27: 47.23 ± 3.80% versus 2.04 ± 0.26%; P < 0.001; CD80: 32.39 ± 5.96,% versus 9.70 ± 2.53%, P < 0.001; CCR7: 33.02 ± 4.03% versus 14.37 ± 1.71%, P < 0.001)  . In contividuals . Altogetin vitro proliferation of γδT cells from patients with sepsis. PBMCs from healthy volunteers responded well to HMBPP plus IL-2/IL-15 with remarkably increased numbers of γδ T cells. In contrast, PBMCs from patients with sepsis failed to respond to HMBPP stimulation . However, no significant changes in the expression of the above molecules were observed in the γδT cells from patients with sepsis upon HMBPP stimulation .As adhesion is considered the first crucial step of antigen presentation , 23, we h sepsis . Howeverthe same . Consequ+ αβ T cells. To validate this, we co-cultured activated γδ T cells with CD4+ αβ T cells. CD4+ αβ T cells showed significant proliferation after 6 days of co-culture with HMBPP-simulated γδ T cells from healthy individuals. In contrast, γδ T cells from patients with sepsis were not able to induce the proliferation of CD4+ αβ T cells  days after organ dysfunction, thus representing relatively late stages of sepsis. Second, the impairment in the antigen-presenting function of γδ T cells in sepsis was reported phenotypically; however, the underlying mechanisms still need to be elucidated. Finally, the antigen-presenting process is quite complex. Therefore, it is difficult to understand the mechanisms involved only using Our study shows that the antigen-presenting function of γδ T cells is severely impaired and may play a role in sepsis-induced immunosuppression.uxab029_suppl_Supplementary_MaterialClick here for additional data file.


Community mobility involves walking with physical and cognitive challenges. In older adults , we assessed gait speed and smoothness (harmonic-ratio) while walking on even and uneven surfaces, with or without an alternate alphabeting dual-task (ABC). ANOVA assessed surface and dual-task effects; Pearson correlations compared gait with global cognition and executive function composite z-scores. The four conditions  affected speed(m/s) (0.97±0.14 vs 0.90±0.15 vs 0.83±0.17 vs 0.79±0.16). Smoothness (2.19±0.48 vs 1.89±0.38 vs 1.92±0.53 vs 1.7±0.43) was affected by only surface (controlled for speed). Greater speed was associated with better global cognition for all conditions and with better executive function for even-ABC and uneven-ABC. Executive function was associated with smoothness during even and uneven walking. Type of walking challenge differentially affects gait quality and associations with cognitive function.


In mammals and yeast, α- and β’-COP WD40 domains mediate cargo-selective interactions with dilysine motifs present in canonical cargoes of COPI vesicles. In contrast to mammals and yeast, three isoforms of β’-COP (β’1-3-COP) have been identified in Arabidopsis. To understand the role of Arabidopsis β’-COP isoforms in plant biology, we have identified and characterized loss-of-function mutants of the three isoforms, and double mutants were also generated. We have found that the trafficking of a canonical dilysine cargo (the p24 family protein p24δ5) is affected in β’-COP double mutants. By western blot analysis, it is also shown that protein levels of α-COP are reduced in the β’-COP double mutants. Although none of the single mutants showed an obvious growth defect, double mutants showed different growth phenotypes. The double mutant analysis suggests that, under standard growth conditions, β’1-COP can compensate for the loss of both β’2-COP and β’3-COP and may have a prominent role during seedling development.Coat protein I (COPI) is necessary for intra-Golgi transport and retrograde transport from the Golgi apparatus back to the endoplasmic reticulum. The key component of the COPI coat is the coatomer complex, which is composed of seven subunits (α/β/β’/γ/δ/ε/ζ) and is recruited  Coat Protein I (COPI)-coated vesicles are involved in transport between Golgi cisternae and in retrograde transport from the Golgi apparatus back to the endoplasmic reticulum (ER) . In mammen bloc from the cytosol onto Golgi membranes [The COPI coat is based on a cytosolic complex (coatomer), containing seven equimolar subunits , which interacts with Golgi membranes via the GTPase ADP-ribosylation factor 1 (ARF1). Cytosolic (GDP-bound) ARF1 first interacts with dimers of p24 family proteins, but following GTP/GDP exchange, ARF1–GTP dissociates from p24 proteins and inserts into Golgi membranes. Coatomer can then interact both with ARF1–GTP and with sorting signals  in the cytosolic domain of p24 family proteins and other COPI cargo proteins. Coatomer polymerization induces COPI vesicle formation, whereas COPI uncoating requires GTP hydrolysis in ARF1 . In contembranes ,6. Howevembranes , and indembranes . Therefoembranes . In the embranes .Cargo recognition by the COPI complex is mediated by sorting signals on the cytosolic domain of transmembrane proteins. The best characterized sorting signals are the dilysine KKxx and KxKxx motifs, present in many ER-resident membrane proteins, including proteins of the p24 family ,14,15,16Saccharomyces cerevisiae. Only ε-COP, emerging last during evolution, is non-essential [in vitro from HeLa cells did not show any significant difference in their cargo content, suggesting that these paralogs may be functionally redundant [In yeast, all COPI subunits are encoded by single genes. Six of the seven subunits are essential for viability in ssential . The COPssential ,22. In mssential . Howeveredundant . Neverthedundant  and, veredundant .Arabidopsis (and most plant species) possesses several isoforms for most COPI subunits. In Arabidopsis, all COPI subunits (except δ- and γ-COP) have two to three isoforms, including α- (α1 and α2) and β’ -COP, with a potential role in cargo binding  [e motif) . Therefoe motif) . Howeverβ’-, γ- and δ-COP caused growth arrest and acute plant cell death in Nicotiana benthamiana, while silencing of β’-COP in tobacco BY-2 cells caused aberrant cell plate formation during cytokinesis [Arabidopsis changed the localization of endomembrane proteins, containing a KXD/E motif involved in COPI binding and Golgi localization [Arabidopsis α1-COP has been shown to be required for the early acceptance of compatible pollen grains [Arabidopsis.Silencing of okinesis . Knockdolization . Arabidon grains  and α2-Cn grains . Besidesn grains ,28,30,31n grains ,33. HereArabidopsis thaliana (ecotype Col-0) was used as wild type. The loss-of-function mutants β’1-cop-1 (SALK_206753), β’1-cop-2 (WiscDsLoxHs036_02G), β’2-cop-1 (SALK_056771), β’3-cop-1 (SALK_004817), β’3-cop-2 (SALK_206870) and β’3-cop-3 (SALK_096549) were from the Salk Institute Genomic Analysis Laboratory and obtained from the Nottingham Arabidopsis Stock Centre. In β’1-cop-1 and β’3-cop-2 mutants, next-generation sequencing detected only one T-DNA insertion. Mutant lines were characterized by PCR  and mutants were sown on Murashige and Skoog (MS) plates containing 135–150 mM NaCl. Plates were transferred to a controlled growth chamber after cold treatment in the dark for three days at 4 °C. After 12 days, the rates of cotyledon greening were scored. To study KCl (100–110 mM) tolerance, the same protocol was used. Seeds harvested from Col-0 and mutant plants grown under the same conditions and at the same time were used.d by PCR . A. thalSEC31A in a total volume of 20 μL. The specificity of the PCR amplification was confirmed with a heat dissociation curve (from 60 to 95 °C). Relative mRNA abundance was calculated using the comparative Ct method, according to Pfaffl [Total RNA was extracted from seedlings using a NucleoSpin RNA plant kit , and 3 μg of the RNA solution were reverse transcribed using the maxima first strand cDNA synthesis kit for quantitative RT-PCR , according to the manufacturer’s instructions. Semi-quantitative PCRs (sqPCRs) were performed on a cDNA template using the PCR Master kit  . The sequences of the primers used for PCR amplifications are included in o Pfaffl . Primersβ’1β’3-cop-1 and β’2β’3-cop-2 plants with the RFP–p24δ5 construct via Agrobacterium using the floral dip method, according to standard procedures [Transgenic plants were generated by transformation of Col-0, ocedures . The RFPocedures ,36. The ocedures . T1 plan®.Imaging was performed using an Olympus FV1000 confocal microscope with a 60× water lens. A fluorescence signal for RFP (543 nm/593–636 nm) was detected. Sequential scanning was used to avoid any interference between fluorescence channels. Post-acquisition image processing was performed using the FV10-ASW4.2 Viewerβ’-COP genes were used with ARES-GT software  [β’2-COP specific sgRNA sequences targeting exons 5 and 7. The CRISPR–Cas9 constructs were designed using the GoldenBraid 3.0 assembly approach  [β’3-cop-2 mutant plants by floral dip for Agrobacterium-mediated transformation [The sequences of the three er 2021)  for seleer 2021) . The gRNer 2021)  .To confirm CRISPR–Cas9-mediated editing of the target gen, young leaves’ genomic DNA was obtained following the protocol described by Edwards  and the g and 4 °C, and the post nuclear supernatant (PNS) was collected. Then, the PNS was centrifuged for 10 min at 1,000,000× g and 4 °C, and the supernatant was collected as a cytosolic extract.Cotyledons of seven-day-old wild type plants and loss-of-function mutants were ground in homogenization buffer  supplemented with 1 mM DTT and a Protease Inhibitor Cocktail , using a mortar and pestle. The homogenate was centrifuged two times for 5 min at 1200× Protein quantitation was performed by using the Bradford assay . Protein extracts were resolved by SDS polyacrylamide gel electrophoresis (SDS–PAGE) and proteins were then transferred to nitrocellulose membranes . Membranes were stained with Ponceau S solution (Sigma) before incubation with primary antibodies against COPI subunits and peroxidase-labeled secondary antibodies. The luminescent signal was developed using the SuperSignal West Pico chemiluminescent substrate . Polyclonal antibodies against mammalian β′-COP (C1PL) and α-COP were kindly provided by Dr F. Wieland . Immunoblots were analyzed using the ChemiDoc XRS + imaging system . Immunoblots in the linear range of detection were quantified using Quantity One software (Bio-Rad Laboratories), with the Ponceau stain protein as a loading control.t-test with unequal variances (Microsoft Excel 2013) among all the β’-COP mutants compared to Col-0.Differences in stress responses, protein levels in western blotting analysis and mRNA levels in RT-sqPCR were tested using a two-sample β’-COP genes were identified in Arabidopsis: β’1-COP (At1g52360), β’2-COP (At3g15980) and β’3-COP (At1g79990). They all encode proteins between 104–105 kDa. β’1-COP and β’2-COP share 88%, β’1-COP and β’3-COP, 85% and β’2-COP and β’3-COP, 81% amino acids, respectively , β’1-cop-1 (SALK_206753) and β’1-cop-2 (WiscDsLoxHs036_02G), were characterized  mutant. Therefore, for the following experiments, we used β’1-cop-1, a β’1-COP mutant with only one T-DNA insertion confirmed by Next-Generation Sequencing (NGS).To gain insight into the physiological role of the three cterized . No β’1-e levels A. These β’2-cop-1 (SALK_056771) is the only T-DNA mutant of β’2-COP found in the SALK collection , β’3-cop-2 (SALK_206870) and β’3-cop-3 (SALK_096549), were characterized , not only a reduction in the expression was detected but also the molecular weight of the band obtained was smaller in the mutant compared to wild type  were due to the presence of truncated transcripts down the T-DNA insertion and showed that this mutant indeed lacked the full length β’3-COP transcript  that may be partially functional.Three cterized . mRNA leectively C. No β’3’3-cop-2 C. When tild type . The seqβ’2-cop and β’3-cop mutants showed any obvious phenotypic alteration under standard growth conditions when compared to wild type plants, and only β’1-cop-1 mutant showed slightly reduced plant height  was used. In addition, β’2β’3-cop-1  was also used to compare with β’1β’3-cop-1 , the only homozygous line obtained for the crosses β’1-cop x β’3-cop. We have shown previously that depletion of β-COP compromises tolerance to NaCl and KCl in Arabidopsis [β’2β’3-cop-2, which has a wild-type phenotype under standard growth conditions, also showed enhanced sensitivity to NaCl and KCl, using the range of NaCl and KCl concentrations used in our previous studies with β-cop mutants [β’2β’3-cop mutants had enhanced sensitivity to NaCl and KCl, and this was also the case for β’1β’3-cop-1. β’2β’3-cop-1, which is a knockdown mutant of β’3-COP, showed less sensitivity to NaCl and KCl than β’2β’3-cop-2 (KO for β’3-COP)  . On the lization ,33. Fina’3-cop-1 C, β’2β’3’3-cop-2 C showed  mutants  as well  mutants  and a qu mutants .Arabidopsis β’-COP antibodies available. As shown in β’1-cop-1, β’3-cop-1 and β’3-cop-2 mutants, while in β’2-cop-1 mutant, only a faint band was detected. The β’-COP antibody also recognized a clear band of approximately 100 kDa in β’1β’3-cop and a faint band in β’2β’3-cop  showed reduced mRNA levels of both α1-COP and α2-COP, although the reduction in α1-COP was much higher than in α2-COP.To check whether the decrease in α-COP protein levels found in the β’-COP double mutants. As shown in β’1β’3-cop and in β’2β’3-cop mutants RFP–p24δ5 showed a predominant localization to the vacuole lumen, with some partial ER localization. This is consistent with impaired retrograde trafficking of p24δ5 from the Golgi back to the ER in the mutants and with previous results showing that transport to the vacuole may be a default pathway for membrane proteins in the plant secretory pathway [β’-COP has been shown to bind to dilysine motifs, which are present in canonical COPI cargoes. One of these cargoes is p24δ5, a protein of the p24 family, which has been previously shown to localize to the ER due to COPI-dependent Golgi-to-ER transport based on a dilysine motif at its C-terminal tail ,50. Ther pathway . This reβ’2-COP in β’2-cop-1 were around 2% of wild type levels . To confirm this, KO β’2-cop mutants were generated by CRISPR/Cas9 gene editing. Two sgRNA sequences were designed using ARES-GT software  targeting exons 5 and 7 of β’2-COP, respectively  have different paralogs in n plants , which mArabidopsis β’-COP isoforms. To this end, we characterized single and double β’-COP mutants. Under standard growth conditions, none of the single β’-COP mutants displayed severe developmental defects, which was likely caused by at least partial functional redundancy among Arabidopsis β’-COP genes. β’-COP double mutant analysis under standard growth conditions suggests that β’3-COP cannot compensate for the simultaneous loss of β’1-COP and β’2-COP. Similarly, β’2-COP cannot compensate for the simultaneous loss of β’1-COP and β’3-COP, as the β’1β’3-cop double mutant failed to develop beyond the seedling stage. However, β’2β’3-cop double mutants had no major phenotypic alterations, indicating that β’1-COP does seem to compensate for the simultaneous lack of β’2-COP and β’3-COP. The results of double mutant analysis appear to correlate with the seed development expression patterns of β’1-COP and suggest a role of β’1-COP during seed development that may affect seedling growth.In this work, we have used a loss-of-function approach to analyze the function of the three en bloc from the cytosol onto Golgi membranes. However, it is not clear how the levels of the different COPI subunits are regulated and how the absence of any of the subunits affects the structure of the complex and the stability of the other subunits. In this work, we found that the protein levels of another COPI subunit, α–COP, was not affected in the single β’-COP mutants. However, β’-COP double mutants showed a dramatic decrease in the levels of α-COP. This is consistent with the known interaction between α- and β’-COP subunits in the B-subcomplex, and suggests that the α–COP subunit is destabilized in the absence of β’-COP. Strikingly, the sec27-1 yeast β’-COP mutant  also showed a reduction in the levels of α-COP [β’1β’3-cop-1, but not β’2β’3-cop-2, have also lower α1/α2-COP mRNA levels than wild-type, which may also contribute to the decrease in α-COP protein levels in this mutant. As α1-COP mRNA levels were more affected than α2-COP mRNA levels in β’1β’3-cop-1, it would be interesting to test in the future whether the isoform α1-COP is a specific partner of β’1-COP. Further experiments should be performed to clarify these issues.Coatomer is made of seven equimolar COPI subunits and is recruited of α-COP . This waβ’-COP mutant [β’1β’3-cop and β’2β’3-cop double mutants showed a mislocalization of p24δ5, which contains a cytosolic C-terminal dilysine motif, from the ER to the vacuole. This may be due to impaired COPI-dependent Golgi-to-ER transport of p24δ5 (The β’-COP subunit has been shown to play a role in binding to dilysine motifs in canonical COPI cargo proteins. Therefore, we hypothesized that loss of β’-COP may affect trafficking of dilysine cargo proteins, as observed in the sec27-1 yeast P mutant . Indeed,of p24δ5 , which iArabidopsis β’-COP proteins are very similar. Residues which have been shown to be important for the interaction of β’-COP with dilysine motifs [Arabidopsis paralogs, and thus one would not expect differential cargo binding among these paralogs. This may explain why β’1β’3-cop and β’2β’3-cop, although they showed different phenotypes under standard growth conditions, both showed mislocalization of p24δ5. The different phenotypes could be explained by different expression patterns of the β’-COP isoforms, as described above. However, it cannot be discarded that the different β’-COP isoforms have subsets of specific cargoes that could be responsible for the different observed phenotypes.The sequences of e motifs  are consAltogether, our findings support an essential role of β’1-COP during seedling development. Future experiments should be performed to determine whether this role is due to its tissue or/and development pattern of expression or to a unique function of the β’1-COP isoform.


Tension regulates α-catenin conformation. Actomyosin-generated force stretches the middle (M)-region to relieve autoinhibition and reveal a binding site for the actin-binding protein vinculin. It is not known whether the intramolecular interactions that regulate epithelial (αE)-catenin binding are conserved across the α-catenin family. Here, we describe the biochemical properties of testes (αT)-catenin, an α-catenin isoform critical for cardiac function and how intramolecular interactions regulate vinculin-binding autoinhibition. Isothermal titration calorimetry showed that αT-catenin binds the β-catenin–N-cadherin complex with a similar low nanomolar affinity to that of αE-catenin. Limited proteolysis revealed that the αT-catenin M-region adopts a more open conformation than αE-catenin. The αT-catenin M-region binds the vinculin N-terminus with low nanomolar affinity, indicating that the isolated αT-catenin M-region is not autoinhibited and thereby distinct from αE-catenin. However, the αT-catenin head (N- and M-regions) binds vinculin 1000-fold more weakly (low micromolar affinity), indicating that the N-terminus regulates the M-region binding to vinculin. In cells, αT-catenin recruitment of vinculin to cell–cell contacts requires the actin-binding domain and actomyosin-generated tension, indicating that force regulates vinculin binding. Together, our results show that the αT-catenin N-terminus is required to maintain M-region autoinhibition and modulate vinculin binding. We postulate that the unique molecular properties of αT-catenin allow it to function as a scaffold for building specific adhesion complexes. Cnt cells , 5, 6. Tnt cells , 12, 13.The best characterized member of the α-catenin family of proteins is mammalian epithelial (αE)-catenin. Structurally, it is composed of 5 four-helix bundles connected to a C-terminal five-helix bundle by a flexible linker , 15, 16.Three α-catenin family proteins are expressed in mammals: the ubiquitous αE-catenin, neuronal (αN)-catenin, and testes (αT)-catenin , 37. αE-Despite a growing awareness of its importance in human disease, the molecular properties and ligand interactions of αT-catenin remain poorly understood. Our previous work revealed that αT-catenin, unlike mammalian αE-catenin, is a monomer in the solution that can bind to F-actin with low micromolar affinity in the absence of tension. F-actin binding is also not allosterically regulated, as the β-catenin–αT-catenin complex binds to F-actin with the same affinity as the αT-catenin monomer . Single-in vitro, revealing that they may compete with one another for binding β-catenin at the plasma membrane. We also show that the M-region of αT-catenin is not autoinhibited and can bind the vinculin N-terminus in the absence of tension with strong affinity. Unlike αE-catenin, however, when the N-terminus of αT-catenin is attached to the M-region, the affinity for vinculin drops significantly. This indicates that interdomain interactions between the N-terminus and the M-region of αT-catenin regulate its interaction with vinculin. We measured the recruitment of vinculin to cell–cell contacts and found that, despite the distinct mechanism of regulation, recruitment of vinculin is still tension dependent. Our findings indicate that the way in which tension regulates vinculin binding differs between αE-catenin and αT-catenin. We postulate that this mechanism is important for the ability of αT-catenin to build specific and distinct molecular complexes at AJs.Here we show that αT-catenin associates with the components of the cadherin–catenin complex with an affinity similar to αE-catenin A) for these experiments because it is more stable than full-length αT-catenin and yields sufficiently high protein concentrations for ITC. Past studies revealed that the αE-catenin head region (aa 1–651) binds β-catenin and the β-catenin–E-cadherin tail complex with a similar affinity to full-length αE-catenin  using purified recombinant proteins. We used the head region (comprising the N- and M-domains) of αT-catenin .Figure cyto) influences the αT-catenin–β-catenin interaction by titrating the β-catenin–Ncadcyto complex into αT-catenin head (C). The affinity of this interaction was 5 to 6 nM  and compared them to the V94D mutant. Analytical size-exclusion chromatography of αT-catenin WT N1-N2 and V94D N1-N2 revealed that WT N1-N2 eluted as a single monomer species , whereas the V94D mutant eluted as a dimer species .Full-length αT-catenin is primarily a monomer in the solution, although it does have homodimerization potential in vitro . The besin vitro , 44. We in vitro A and comB). Incubation with BS3 did not affect migration at low concentrations, although at higher concentrations (4 and 8 μM), larger species were detected at both temperatures. In contrast, αT-catenin V94D N1-N2 ran as 50-kDa protein in the presence of BS3 at all concentrations tested (C), indicating a cross-linked dimer. We conclude that the αT-catenin N terminus, similar to full-length protein, is primarily a monomer in the solution.We then analyzed the oligomeric state of the αT-catenin N-terminus by cross-linking. Increasing concentrations of αT-catenin N1-N2 were incubated with or without the cross-linker bis(sulfosuccinimidyl)suberate (BS3) at 4 °C or 37 °C and the resulting products analyzed by SDS-PAGE. As expected, the αT-catenin N1-N2 migrated as a 25-kDa protein in the absence of a cross-linker B. Incubas tested C, indicacyto complex by ITC. The affinity of this interaction was 7 nM . We then probed the M-region flexibility by limited trypsin proteolysis. Tryptic digestion of αE-catenin M1-M3 revealed that it was largely protease resistant: nearly 50% of the fragment was still intact after 120 min of digestion, consistent with it forming a closed, autoinhibited domain . In contrast, αT-catenin M1-M3 was completely digested after 60 min into three stable fragments at 23, 16, and 12 kDa . Note that both M regions contain a similar number of lysine and arginine residues (47 in αE-catenin and 39 in αT-catenin), the majority of which are conserved. N-terminal sequencing revealed that the 23-kDa and 16-kDa fragments both started at aa 379 and represented the M2-M3 and M2 bundles, respectively. The 12-kDa fragment started at aa 485 and corresponded to the M3 bundle . Similar protease-resistant fragments were observed from digest of full-length protein . αT-catenin M1-M3 eluted as a single, discrete peak by size-exclusion chromatography, identical to αE-catenin M1-M3  that contains the first 2 four-helix bundles and binds to αE-catenin with a similar affinity to the vinculin head domain, D1-D4 . As obseFig. 3in vitro single-molecule stretching experiments revealed that force is required to expose the vinculin-binding site in αT-catenin M1-M3  did not weaken vinculin D1 binding relative to M1-M3 . αE-catenin and αT-catenin recruited similar levels of vinculin in dimethyl sulfoxide controls . Importantly, blebbistatin treatment significantly reduced vinculin recruitment to both αE-catenin and αT-catenin containing AJs (C–E). Thus, myosin-based tension is required to recruit vinculin to αT-catenin, similar to αE-catenin.We tested the ability of αT-catenin to restore cell–cell adhesion and recruit vinculin in α-catenin-deficient R2/7 carcinoma cells . We tranning AJs , C–E. ThWe then examined the correlation between EGFP fusion expression and vinculin recruitment. As expected, EGFP–αE-catenin expression levels did not correlate with vinculin levels because vinculin was only recruited when αE-catenin was activated by sufficient tension , 28. HowN). αE-catenin and αT-catenin N1-M3  were both cytosolic and, as expected, unable to form cell–cell contacts and recruit vinculin (J and K). Deletion of M3 in αE-catenin relieves autoinhibition, and αE-catenin N1-M2 (aa 1–510) was able to recruit vinculin and restore cell–cell adhesion, as shown previously  or the head domain minus M3 and ABD (N1-M2). All EGFP-tagged αE-catenin and αT-catenin fragments expressed as soluble proteins of the predicted size N. αE-catvinculin , J and Keviously . Here vicyto complex binds with high, ∼5-nM affinity to αT-catenin similar to 1- to 2-nM affinity previously reported between the β-catenin–cadherin tail complex and αE-catenin . This suggest that αT-catenin does not compete with αE-catenin for binding cytosolic β-catenin. Although there is evidence of a cytosolic αE-catenin–β-catenin complex in epithelial cells  between the αT-catenin N-terminus and M-region promote M1 folding and M-region autoinhibition. Interestingly, the first 56 residues of αE-catenin contribute to the inhibition of vinculin binding in addition to the M3 domain (The αT-catenin head fragment (N1-M3) showed weak, micromolar binding to vinculin, indicating that the N-terminus regulates M-region autoinhibition. The entire N-terminus (N1-N2) is required for autoinhibition because deleting N1 restored strong vinculin binding. Notably, replacing the N-terminus with another well-structured protein moiety, the titin I27 repeats, reduced vinculin binding and caused the binding reaction to switch from exothermic to endothermic, suggesting the M1 was stabilized. This observation explains the force-dependent vinculin binding to the αT-catenin M-region observed in recent biophysical experiments . The abi3 domain .Expression of EGFP–αT-catenin was sufficient to restore cell–cell contacts and recruit vinculin in α-catenin-deficient R2/7 cells. Vinculin binding to αT-catenin requires actin binding by the latter and is tension dependent, similar to αE-catenin. This is consistent with the vinculin-binding site in the αT-catenin M1 domain being occluded in the absence of force. Notably, deletion of M3 in αT-catenin did not relieve autoinhibition, offering additional evidence that the intramolecular interactions required for autoinhibition in αT-catenin differ from αE-catenin.in vivo. Loss of αE-catenin from the mouse heart disrupts AJ formation and causes a marked decrease in vinculin expression and recruitment, despite the presence of αT-catenin, resulting in cardiomyopathy  in pGEX-TEV were described previously (M. musculus αT-catenin N1-N2 (aa 1–259), N1-M3 (aa 1–659), M1-M3 (aa 260–626), N2-M3 (aa 147–626), and αE-catenin M1-M3 (aa 273–651) and M1-M2 (aa 273–510) were cloned into pGEX-TEV. The construct encoding αT-catenin M1-M3 flanked by I27 handles in pET151/D-TOPO was kindly provided to us by Jie Yan  in 0.05 mg/ml sequencing grade trypsin (Roche Applied Science). Digestions were stopped with 2X Laemmli sample buffer and placed on ice until analysis. Samples were boiled and run by SDS-PAGE and then stained with 0.1% Coomassie Brilliant Blue R-250, 40% ethanol, and 10% acetic acid. Gels were scanned on a LI-COR scanner. For N-terminal sequencing, digested peptides were blotted onto polyvinylidene difluoride membrane, stained , destained, and dried. Individual bands were excised from the membrane and sequenced by Edman degradation (Iowa State University Protein Facility).αT-catenin protein fragments were incubated with or without 1-mM BS3 (Thermo Scientific) in 20-mM Hepes, pH 7.4, 150-mM NaCl, and 1-mM DTT for 30 min at 4 °C or 37 °C, separated by SDS-PAGE, stained with the Coomassie dye, and imaged on a LI-COR scanner.cyto complex, or 396- to 600-μM vinculin aa 1 to 259 (D1) into the cell containing 9- to 55-μM of αT-catenin or αE-catenin. For experiments on the MicroCal VP-ITC, the ligand was added with an initial 2-μl injection followed by 32 × 9-μl injections. The concentration of αT-catenin head or αT-catenin head–β-catenin complex in the cell varied between 22 and 56 μM. Vinculin D1 concentrations in the syringe varied between 240 and 546 μM. All calorimetry experiments were performed at 25 °C. All data analyses were performed using Malvern MicroCal ITC analysis software. For baseline correction, a mean baseline value, calculated by averaging the data points at saturation, was subtracted from each data point.Proteins used for ITC were purified as described except the S200 buffer was replaced with the ITC buffer . An identical buffer was used to purify both cell and titrant samples to ensure buffer match. Only fresh, unfrozen proteins were used for ITC. Measurements were performed on a Malvern MicroCal PEAQ-ITC or MicroCal VP-ITC calorimeter . For experiments on the MicroCal PEAQ-ITC, titration occurred by an initial 0.5-μl injection followed by 18 × 2-μl injections of 110- to 150-μM β-catenin, 103- to 158-μM β-catenin–NcadR2/7 carcinoma cells were cultured in Dulbecco's modified Eagle's medium (4.5 g/l glucose), 10% fetal bovine serum, 1-mM sodium pyruvate, and penicillin/streptomycin. Lipofectamine 2000 was used for all transient transfections.R2/7 cells were lysed 48 to 72 h after transfection in RIPA buffer  plus protease inhibitors (Millipore). The lysate protein concentration was measured by bicinchoninic acid protein assay, and 15 μg of each sample was separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membrane was blocked in TBST  + 5% BSA  for 1 h at RT, washed in TBST, and incubated with anti-GFP  and anti-GAPDH  antibodies for 1 h at RT. The membrane was washed twice in TBST and then incubated with anti-rabbit IRDye 680 and anti-mouse IRDye 800 for 1 h at RT, washed twice with TBST, and washed once with PBS. Membranes were scanned on a Bio-Rad ChemiDoc MP imaging system.Cells were fixed in 4% paraformaldehyde in PHEM buffer , washed with PBS, permeabilized with 0.2% Triton X-100 in PBS for 2 min, and then blocked for 1 h at RT in PBS +10% BSA. Samples were washed three times in PBS, incubated with the primary antibody in PBS +1% BSA for 1 h at RT, washed three times in PBS, incubated with the secondary antibody in PBS +1% BSA for 1 h at RT, washed three times in PBS, and mounted on the ProLong Diamond mounting medium. Cells were imaged on a Nikon Eclipse Ti inverted microscope outfitted with a Prairie swept-field confocal scanner, Agilent monolithic laser launch, and Andor iXon3 camera using NIS-Elements imaging software.p < 0.05.To quantify vinculin recruitment to EGFP-tagged αE-catenin and αT-catenin, a maximum projection was created from four planes of the z-stack  where cell–cell contacts were best in focus. IsoJ Dark thresholding was used to create a mask of the GFP channel to define the region of analysis in ImageJ. Vinculin signal intensity was then measured within the masked region. Next, three random intensity measurements of vinculin staining were taken in the cell cytoplasm and these values averaged. Finally, the vinculin intensity within the mask was divided by the cytoplasmic signal to normalize between samples and calculate the contact/cytoplasmic ratio. Colocalization data were plotted with Prism software (GraphPad). A one-way ANOVA with Tukey’s comparisons was performed to determine significance; 2 value, and p value using Prism software (GraphPad).To examine the relationship between EGPF–αE-catenin or EGFP–αT-catenin levels and vinculin recruitment, the vinculin contact/cytoplasmic ratio was plotted against the average GFP intensity of each fusion construct within the masked cell contact region (described above). Linear regression analysis was performed to calculate the slope, 95% confidence intervals, RAll data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


In this article , the autIn the methods section, “LAG-3 ” should be changed to “LAG-3 ”.In the Table 2 section, the title “Expression of LAG-3, PD-1, and PD-L1, CD3, GZMB in NSCLC patients” should be changed to “Expression of LAG-3, PD-1, and PD-L1, CD3, GZMB in NPC patients”. Correspondingly, “NSCLC: non-small cell lung cancer” should be changed to “NPC: nasopharyngeal carcinoma” in the caption of the table.


Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) revealed their key role in immune regulation, offering promising therapeutic perspectives for immune and inflammatory diseases. We aimed to develop a production process of an UC-MSC-based product and then to characterize UC-MSC properties and immunomodulatory activities in vitro, related to their clinical use and finally, to transfer this technology to a good manufacturing practice (GMP) compliant facility, to manufacture an advanced therapy medicinal product (ATMP).Fifteen human umbilical cords (UCs) were collected to develop the production process. Three batches of UC-MSCs from a single donor were characterized at basal state and after in vitro pro-inflammatory stimulation by interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα). Proliferation, immunophenotype, activation markers’ expression and the inhibition of T cell proliferation were assessed. Finally, this technology was transferred to a GMP-compliant facility to manufacture an UC-MSC-based ATMP, from a single donor, using the explant method followed by the establishment of master and work cell stocks.versus non-treated cells. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression was induced significantly after priming. T cell proliferation was significantly decreased in the presence of UC-MSCs in a dose-dependent manner. This inhibitory effect was improved by IFNγ or IFNγ + TNFα, at UC-MSCs:PBMC ratio 1:10 and 1:30, whereas only IFNγ allowed to decrease significantly T cell proliferation at ratio 1:100. The manufacturing process of the UC-MSC-based ATMP was qualified and authorized by the French regulatory agency for clinical use (NCT04333368).Twelve UCs were processed successfully allowing to isolate UC-MSCs with doubling time and population doubling remaining stable until passage 4. CD90, CD105, CD73, CD44, CD29, CD166 expression was positive; CD14, CD45, CD31, HLA-DR, CD40, CD80 and CD86 expression was negative, while CD146 and HLA-ABC expression was heterogeneous. Cell morphology, proliferation and immunophenotype were not modified by inflammatory treatment. Indoleamine 2,3-dioxygenase (IDO) expression was significantly induced by IFNγ and IFNγ + TNFα This work allowed to develop an investigational UC-MSC-based ATMP authorized for clinical use. Our results showed that an inflammatory environment preserves the biological properties of UC-MSCs with an improvement of their immunomodulatory functions.The online version contains supplementary material available at 10.1186/s13287-021-02637-7. A minimum of 95% confidence interval was established for significance. A Before considering the use of UC-MSCs for clinical indications, it was necessary to design a process allowing to isolate and expand the MSCs collected from human UC units as an infinite and easily accessible source of the starting material.Among fifteen collected UCs, twelve (80.0%) were processed successfully, allowing to isolate UC-MSCs. There were three failures to process UC-MSCs, of which two 13.3%) were due to cell isolation failure and one (6.7%) to bacterial contamination during the culture , which was not correlated to the age of donors . We defined this step as passage 0 (P0). After expansion at P1, total cell quantity was 6.6 × 107 [0.7 × 107 − 21.0 × 107]; DT (h) and PD between P0 and P1 were 18.2 [14.3 − 24.4] and 8.1 [5.9 − 10.5], respectively. Cell quantity, DT and PD are described for each UC in Fig. The mean total UC-MSCs quantity isolated per UC was 3.6 × 10n = 4). DT and PD were stable until P4 and were, respectively, 18.4 ± 4.2 h and 7.0 ± 2.5 at P2, 19.5 ± 4.6 h and 6.8 ± 2.2 at P3, 19.2 ± 3.0 h and 7.9 ± 1.5 at P4. From P5, DT increased and were more variable, whereas PD decreased yielding values of 24.1 ± 6.3 h and 6.9 ± 1.3 at P5, 27.5 ± 12.5 h and 6.3 ± 1.8 at P6, 39.2 ± 27.6 h and 5.8 ± 2.0 at P7, respectively . DT (h) and PD were similar at P3 , whereas PD was significantly higher  , a comparison with the standard basal medium minimum essential medium alpha (MEM-α) with 5% PL (MEM-α + 5% PL) was performed. Considering the data above, this analysis was focused on P3 and P4 , and adhesion molecules CD44, CD29 and CD166   by UC-MSCs; conversely, the cells did not express HLA-DR class II (0.1 ± 0.1%) or co-stimulatory molecules CD40, CD80 and CD86  . In addition, no significant difference was observed in the tested cell markers , thereby showing that a simulated inflammatory environment does not affect the UC-MSCs’ immunophenotype  underlying the key role of IFNγ to induce IDO expression.To evaluate UC-MSCs’ immunomodulatory properties, we first assessed the IDO expression, a key molecule involved in the inhibition of T cell proliferation . IDO expp < 0.05, Kruskal–Wallis test, Fig. As MSCs’ immunomodulatory functions involve cell–cell interactions, we assessed the expression of cell surface markers such as immune and adhesion molecules as well as cytokine receptors including ICAM-1/CD54, PD-L1/CD274, VCAM-1/CD106, CD200, IFNγ-R/CD119 and TNFα-RII/CD120b. Our results showed that UC-MSCs expressed moderately ICAM-1/CD54 (70.1 ± 7.6%) and IFNγ-R/CD119 (42.7 ± 15.1%) at basal state , 15.7% (p < 0.0001) and 55.1% (p < 0.01), respectively; for TNFα: 40.8% (p < 0.0001), 46.7% (p < 0.0001) and 63.3% (p < 0.05), respectively; and for IFNγ + TNFα: 3.9% (p < 0.0001), 13.5% (p < 0.0001) and 60.3% (p < 0.05), respectively.To confirm the immunomodulatory properties of UC-MSCs, we performed a potency assay using the MLR assay according to ISCT recommendations , at basap < 0.01 and p < 0.0001) and 1:30 ratios (p < 0.001 for both), whereas only IFNγ allowed to decrease significantly T-Ly proliferation at the 1:100 ratio (p < 0.05)  regardless of the UC-MSCs:PBMCs ratio.Interestingly, T-Ly proliferation was significantly decreased after priming by IFNγ or IFNγ + TNFα compared to basal state, at 1:10  was 23.8 ± 6.4 and the cumulative PD was 25  and WCS (n = 3 batches) and a MLR assay for those of the WCS (n = 3 batches). Viability was 94.0 ± 4.2% for MCS and 91.0 ± 1.6% for WCS and was compliant for all batches (≥ 80%). CD90, CD73 and CD105 markers’ expression was ≥ 90%, whereas CD45, CD34, CD11b, CD19 and HLA-DR was ≤ 2%, according to the defined specifications , supporting the higher immunomodulatory properties of UC-MSCs in vitro at basal state , MCS production, WCS manufacturing, ATMP manufacturing. P: passage, d: day.Additional file 5. Negative controls of CD90, CD105, CD73, CD44, CD29, CD166, CD146, HLA-DR, HLA-ABC, CD40, CD80, CD86 markers expression. Fluorescence Minus One (FMO) expression of CD14, CD45, CD31 markers.Additional file 6. Area under the curve (AUC) representing the UC-MSCs and BM-MSCs’ abilities to inhibit T-Lymphocyte proliferation, which allow to assess their immunomodulatory activity. P < 0.001, Welch’s test.


The three-dimensional supramolecular architecture of the lattice is formed by weak C—H⋯C/S/N hydrogen bonds.The title charge-neutral complex shows a  II(NCS)2(C19H32N8)], consists of two charge-neutral complex molecules. In the complex molecule, the tetradentate ligand N1,N3-bis[methylene]-2,2-dimethylpropane-1,3-diamine coordinates to the FeII ion through the N atoms of the 1,2,3-triazole and aldimine groups. Two thiocyanate anions, also coordinated through their N atoms, complete the coordination sphere of the central Fe ion. In the crystal, neighbouring molecules are linked through weak C—H⋯C/S/N interactions into a three-dimensional network. The intermolecular contacts were quantified using Hirshfeld surface analysis and two-dimensional fingerprint plots, revealing the relative contributions of the contacts to the crystal packing to be H⋯H 50.8%, H⋯C/C⋯H 14.3%, H⋯S/S⋯H 20.5% and H⋯N/N⋯H 12.1%. The average Fe—N bond distance is 2.170 Å, indicating the high-spin state of the FeII ion, which does not change upon cooling, as demonstrated by low-temperature magnetic susceptibility measurements. DFT calculations of energy frameworks at the B3LYP/6–31 G theory level were performed to account for the interactions involved in the crystal structure.The unit cell of the title compound, [Fe II complexes based on Schiff bases derived from N-substituted 1,2,3-triazole aldehydes methylene]-2,2-dimethylpropane-1,3-diamine, and report below structural and magnetic investigations of a new complex with R = tert-butyl.Having interest in functional 3II ion of the title complex has a distorted trigonal–prismatic N6 coordination environment formed by the four N atoms of the tetradentate Schiff-base ligand and the two NCS− counter-ions °. The average trigonal distortion parameters, Σ = Σ112(|90 – iφ|), where φi is the angle N—Fe—N′ , where iθ is the angle generated by superposition of two opposite faces of an octahedron , and are significantly larger than those of similar [FeN6] high-spin trans-complexes . The volume of the [FeN6] coordination polyhedron is 12.60 Å3.The Fens Fig. 1. The aveX hydrogen bonds  to 1.9978 (blue) a.u. The pale-red spots symbolize short contacts and negative dnorm values on the surface correspond to the interactions described above. The overall two-dimensional fingerprint plot is illustrated in Fig. 3dnorm are shown for the H⋯H, H⋯C/C⋯H, H⋯S/S⋯H, and H⋯N/N⋯H contacts, and the two-dimensional fingerprint plots are presented in Fig. 4Hirshfeld surface analysis was performed and the associated two-dimensional fingerprint plots were generated using Eele), the dispersion forces (Edis) and the total energy diagrams (Etot), are shown in Fig. 5a. The cylindrical radii, adjusted to the same scale factor of 80, are proportional to the relative strength of the corresponding energies , reflecting dipole–dipole interactions of the asymmetric complex cis-molecules in the lattice. According to the calculations, the most repulsive interaction is due to the anion-to-anion alignment of neighbouring complex molecules (Etot = 65.3 kJ mol−1) while the ligand-to-anion alignment gives the most attractive one (Etot = −223.9 kJ mol−1) . The colour-coded interaction mappings within a radius of 3.8 Å of a central reference molecule for the title compound together with full details of the various contributions to the total energy (Etot) are given in the Supporting Information.The energy frameworks, calculated using the wave function at the B3LYP/6-3G level of theory for the title compound, including the electrostatic potential forces (1) Fig. 5b. The cχMT versus T (χM is the molar magnetic susceptibility and T is the temperature). At 300 K, the χMT value is close to 3.51 cm3 K mol−1, and on cooling the value remains constant down to 30 K. The decrease of χMT below 30 K is attributed to the zero-field splitting of the high-spin (S = 2) FeII centres  of the title compound using a Quantum Design MPMS2 superconducting quantum interference device (SQUID) susceptometer operating at 1 T. Experimental susceptibilities were corrected for the diamagnetism of the holder (gelatine capsule) and of the constituent atoms by the application of Pascal’s constants. The magnetic behaviour of the compound is shown in Fig. 6et al., 2016II thiocyanate complexes, derivatives of a 1,3-diamine and N-substituted 1,2,3-triazole aldehydes: DURXEV, ADAQUU, ADAREF and solvatomorphs ADAROP and ADARUV  with 1-tert-butyl-1H-1,2,3-triazole-4-carbaldehyde  in boiling methanol (5 ml) over 5 min and subsequently reacted with [Fe(py)4(NCS)2]  and ascorbic acid  in boiling methanol (5 ml). The formed yellow solution was slowly cooled to ambient temperature. Yellow–orange crystals then precipitated and were subsequently filtered off. Elemental analysis calculated (%) for C21H32FeN10S2: C, 46.32; H, 5.92; N, 25.72; S, 11.78. Found: C, 46.40; H, 6.10; N, 26.18; S, 11.80. IR v : 1611 (C=N), 2071, 2116 (NCS).The synthesis of the title compound is identical to that reported by us recently for similar thiocyanate complexes (Znovjyak Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021004412/dj2026sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021004412/dj2026Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021004412/dj2026Isup3.cdxSupporting information file. DOI: 10.1107/S2056989021004412/dj2026sup4.pdfThe full colour-coded interaction mappings of a central reference molecule for the title compound. DOI: 2079827CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Schistosoma. For the last 40 years, control of schistosomiasis in endemic regions has predominantly been facilitated by administration of a single drug, praziquantel. Due to limitations in this mono-chemotherapeutic approach for sustaining schistosomiasis control into the future, alternative anti-schistosomal compounds are increasingly being sought by the drug discovery community. Herein, we describe a multi-pronged, integrated strategy that led to the identification and further exploration of the quinoxaline core as a promising anti-schistosomal scaffold.Schistosomiasis is a neglected disease of poverty that is caused by infection with blood fluke species contained within the genus Schistosoma mansoni . Secondary exploration of the chemical space around compound 1 led to the identification of a quinoxaline-core containing, non-genotoxic lead (compound 22). Compound 22 demonstrated substantially improved activities on both intra-mammalian  and intra-molluscan (sporocyst) S. mansoni lifecycle stages. Further medicinal chemistry optimisation of compound 22, resulting in the generation of 20 additional analogues, improved our understanding of the structure-activity relationship and resulted in considerable improvements in both anti-schistosome potency and selectivity . Some derivatives of compound 22 (e.g. 31 and 33) also demonstrated significant activity against the two other medically important species, Schistosoma haematobium and Schistosoma japonicum. Further optimisation of this class of anti-schistosomal is ongoing and could lead to the development of an urgently needed alternative to praziquantel for assisting in schistosomiasis elimination strategies.Firstly, phenotypic screening of commercially available small molecules resulted in the identification of a moderately active hit compound against   •50 of 0.44 µM and 84.7 nM for schistosomula and adult worms.Lead compound 22 was identified with EC•20 analogues of the lead compound 22 were synthesised.•Compounds 25, 30 and 32 showed the best selectivity profile.•Compounds 31 and 33 are the most active on three medically important schistosome species. The spectral data are in accordance with those reported in the literature +.2, N3-bis-6-(pyrrolidin-1-yl)quinoxaline-2,3-diamine (22b): To a mixture of compound 22b  in CH3CN (2 ml), dibromobutane  and anhydrous K2CO3  was added. The reaction mixture was heated at 150 °C for 15 min. After reaction completion, the mixture was filtered with EtOAc (20 ml). The resultant organic phase was washed with saturated aqueous NaHCO3 (3 × 5 ml) and brine (1 × 10 ml). The organic layer then dried over anhydrous Na2SO4, filtered and reduced to dryness to give a crude product. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 80:20 v/v in 10 CV. The final product 22b was obtained at 28% yield as a brown powder. 1H NMR : δ 9.09 , 8.93 , 8.23 , 8.15 , 7.90 , 7.75 , 7.63 , 7.58 , 7.47 , 6.90 , 6.57 , 3.35 , 2.04–1.96 ; 13C NMR  δ 145.75 (ArC), 141.34 (ArC), 141.20 (ArC), 140.63 (ArC), 138.11 (ArC), 136.89 (ArC), 131.13 (ArC), 131.04 (ArC), 130.72 (ArCH), 130.58 (ArCH), 128.61(ArC), 126.51 (ArCH), 124.09 (ArC), 122.91 (ArC), 121.69 (ArCH), 120.62 (ArCH), 120.43 (ArCH), 119.49 (ArCH), 114.87 (ArCH), 105.12 (ArCH), 48.97 (2 x CH2), 25.01 (2 x CH2); UPLC-MS: Rt: 2.71 min, MS (ESI)+: 518.05 [M − H]+.N5.1.23). The organic phase was washed first with saturated aqueous NaHCO3 (3 × 5 ml) and brine (1 × 10 ml). The organic layer was then dried over anhydrous Na2SO4, filtered and reduced to dryness to give a crude product. The crude product was purified by flash chromatography eluting with n-hexane/EtOAc in different proportions depending on the specific analogue being synthesised.To a solution of compound 22a (1 eq) dissolved in anhydrous DCM, anhydrous pyridine (3.6 eq) was added under nitrogen. The appropriate acyl derivate (1.1 eq) was added dropwise to the above solution cooled to 0 °C in an ice-bath under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 h. After completion, the reaction mixture was diluted with DCM and quenched with saturated aqueous sodium bicarbonate amino)quinoxalin-6-yl)acetamide (22c): pale-yellow powder, 52% yield. 1H NMR : δ 10.11  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O), 9.23 , 9.15 , 8.31 , 8.24 , 8.00 , 7.81 , 7.63 , 7.58–7.50 , 2.09 ; 13C NMR : δ 168.38 (CO), 141.15 (ArC), 140.64 (ArC), 140.43 (ArC), 139.82 (ArC), 137.46 (ArC), 136.30 (ArC), 132.18 (ArC), 130.79 (2 x ArC), 130.47 (ArCH), 130.43 (ArCH), 125.78 (ArCH), 123.52 (ArC), 123.20 (ArC), 121.04 (ArCH), 120.78 (ArCH), 120.05 (ArCH), 119.81 (ArCH), 118.78 (ArCH), 113.96 (ArCH), 24.12 (CH3); UPLC-MS: Rt: 2.19 min, MS (ESI)+: 508.02 [M+H]+. The spectral data are in accordance with those reported in the literature [terature .5.25.2.1S. mansoni) adhered to the United Kingdom Home Office Animals (Scientific Procedures) Act of 1986 (project license PPL 40/3700 and P3B8C46FD) as well as the European Union Animals Directive 2010/63/EU and were approved by Aberystwyth University's Animal Welfare and Ethical Review Body (AWERB). The use of hamsters and mice  was approved by the Institutional Animal Care and Use Committee of the University of California San Diego.All procedures performed on mice  at a stock concentration of 10 mM and a working concentration of 1.6 mM . Small aliquots of both stock and working solutions were stored at −20 °C prior to use. Similar preparations were followed for auranofin  and praziquantel  positive controls used in the schistosomula and adult worm screens, respectively.5.2.3S. mansoni were shed from two Biomphalaria glabrata strains, the NMRI albino and pigmented outbred strains, under intensified lighting conditions (1 h of incubation at 26 °C) [Cercariae derived from the NMRI  Puerto Rican strain (PR-1) of t 26 °C) . Cercarit 26 °C) .2. Following the co-culture period, automatic assessment of compound-induced schistosomula motility and phenotypic changes were conducted with the high-content imaging platform Roboworm using the image analysis model previously described by Paveley et al. [High-throughput screening (HTS) of selected compounds was conducted as previously described ,55. Briey et al. .5.2.4Mus musculus HsdOLa:TO - Tuck Ordinary; Envigo, UK) were used for the mammalian-specific propagation of the S. mansoni life cycle. Experimental animals were infected with 4000 or 180 S. mansoni cercariae/mouse and perfused three- or seven-weeks post-infection to obtain S. mansoni juvenile or adult worms, respectively [l-glutamine  and an antibiotic mixture ) to remove residual host contamination [2 at 37 °C for at least 1 h.Mice  [Juvenile worms were distributed in a 96-well tissue culture plate  containing 200 μl/well. Compounds were added to each treatment well at a concentration range of 10–0.156 μM. Negative (1.25% DMSO) and positive (15 μM PZQ and 15 μM AUR in 1.25% DMSO) controls were also included in each drug screen. Treated juvenile worms were incubated for 72 h in a humidified environment containing 5% COovement) . Additioovement) ,59. In b2 at 37 °C. Parasite motility after compound treatment was assessed by a digital image processing-based system (WormassayGP2) [For the evaluation of anti-schistosomal activity on the adult stage of the parasite, 7-wk old worms were transferred into 48 well tissue culture plates . The worms were dosed with compounds at final concentrations spanning 0.78–50 μM (in 0.5% DMSO). DMSO (0.5%) and praziquantel (10 μM in 0.5% DMSO) were also included as negative and positive control treatments. Treated adult worms were incubated for 72 h in a humidified environment containing 5% COssayGP2) , modifiessayGP2) ,62. Foll5.2.5in vitro miracidia to sporocyst screens, as previously described [Following hepatic portal vein perfusion of TO mice, experimentally infected 7 weeks previously, the mouse livers were collected to isolate parasite eggs. Briefly, the infected tissues were homogenised in double saline solution (1.7% w/v NaCl) using a Waring blender. The homogenates were passed through a 0.45 μm filter to retain egg material and the resulting filtered solution was collected in a volumetric flask. Miracidia hatching was induced by exposure of eggs to light in artificial river water (e.g. Lepple water) ,63,64. Tescribed . Brieflyescribed .5.2.6S. haematobium worms (Egyptian isolate) were recovered from male Golden Syrian hamsters infected with 600 cercariae 20 weeks earlier. Adult S. japonicum worms (Philippine isolate) were recovered from female Swiss Webster mice infected with 100 cercariae five weeks earlier.Adult 2 environment. The next morning, a final concentration of 100 nM compound (in 1 μL DMSO) was added to each well and the final volume made up to 2 ml with Basch 169 medium supplemented with antibiotics and FBS. Each compound was tested in triplicate (except for some instances tested in duplicate). Incubations were maintained at 37 °C in a 5% CO2 environment and phenotypic changes recorded at 5, 24, 48 and 72 h.For each species, parasite recovery involved reverse perfusion of the hepatic portal and mesenteric system ,68 usingUsing a Zeiss Axiovert A1 inverted microscope, the male parasite's phenotypic changes in shape, density and motility were recorded using a constrained nomenclature of simple descriptors ,70,71. T5.32. Cytotoxicity was evaluated by way of a 3--2,5-diphenyl tetrazolium bromide (MTT) assay as previously described [4 cells/well and incubated in an atmosphere of 5% CO2 at 37 °C. Following a 24 h incubation, cells were incubated with the compounds at seven different concentrations  for a further 24 h in a CO2 incubator at 37 °C. Alternatively, when a lower seeding density (5 × 103 cells/well) was used, cells were dosed at 24 h and incubated in a CO2 incubator at 37 °C for 72 h (the incubation time used for both schistosomula and adult worm screens). In both cases, control cell treatments included DMSO  and Triton X-100 . Each compound was tested in triplicate and each experiment was performed either three (for 24 h timepoint) or two times (for 72 h timepoint). The absorbance readings (at 570 nm) of the soluble formazan crystals were recorded using PolarStar Omega plate reader (BMG-Labtech). Dose response curves were generated by non-linear regression analysis in GraphPad Prism 7.02 , after log concentration transformation and normalization of the absorbance readings of each concentration point. The cytotoxic concentration inhibiting 50% cell growth (CC50) was derived at 24 and 72 h. The latter was used to calculate the selectivity index (SI) of the compounds presented herein.The Human Caucasian Hepatocyte Carcinoma (HepG2) cell line  was used to perform cytotoxicity tests of compounds used in this study. Initially the cells were handled under sterile conditions and grown at 37 °C in a humidified atmosphere containing 5% COescribed . The cel5.4in vitro micronucleus (MN) assay was employed for the assessment of chromosome damage. Human, p53 competent, lymphoblastoid TK6 cells (Cat. No. 95111735) were used in this study and obtained from the European Collection of Authenticated Cell Cultures (ECACC) [l-glutamine  and hygromycine-B was used for TK6 cell culture [The  (ECACC) ). RPMI 1 culture . Cells w5 TK6 cells/ml were placed in a series of sterile vented tissue culture flasks (Fisher brand) and treated for 1.5–2 cell-cycle period (24 h) with no recovery with compound 22, the negative control (1% DMSO) or the positive controls (45.4 μM Methyl methanesulphonate (MMS) and 8.4 μM Carbendazim (Crbz) in DMSO; supplied from Sigma-Aldrich - CAS numbers 66-27-3 and 10605-21-7, respectively). All incubation steps occurred at 37 °C in a humidified atmosphere containing 5% CO2.Once cells reached confluency, sub-cultures were established. Approximately 2 × 10g for 5 min at room temperature, the supernatant was discarded, and the pellet re-suspended in pre-warmed phosphate-buffered saline (PBS). Subsequently, the PBS was removed via centrifugation at 200×g and the pellet was re-suspended in BD FACS™ lysis solution (CAS 349202) enabling cell fixation and membrane permeabilization. Post fixation, cells were centrifuged for 5 min at 200×g, supernatant discarded and subsequently stained for ɣH2AX (using Alexa Fluor® 488 Mouse anti-H2AX (pS139), Clone N1-431, Mouse BALB/c IgG1, κ, Cat. No. 560445, BD Biosciences) and p53 , as described [After the treatment period, cultures were centrifuged at 200×escribed . FollowiFor MNi assessment, of the 14,000–15,000 viable cells per experiment, about 4000 cell images were assessed except the positive control (Crbz) where 1600 cells were examined. Data analysis was performed as described . BrieflyThe full 14,000–15,000 cell population was assessed for the biomarkers ɣH2AX and p53 alongside compound 22 autofluorescence. Gating parameters for ɣH2AX and p53 were determined based on the cell populations of unstained PBS controls and stained DMSO controls compared to positive control samples. These same gates were then applied for the batch processing of 22-treated cell populations.Percentage of cells identified at each fluorescence channel were recorded in three independent experiments and data were then transformed as fold changes. A cut off for a negative vs a positive ɣH2AX and p53 response over the control is defined as a 1.5-fold change ,37,39. T5.5To investigate the compound fluorescence emission, a preliminary lambda scan was performed on HeLa cells treated for 6 h with 5 μM of compound 22 in DMEM (Fisher Scientific) containing 10% FBS. Cells were washed three times in phenol red free DMEM and imaged using a Leica SP5 laser scanning confocal microscope.Fluorescence intensity was measured following 405 nm excitation by sequentially imaging at 9 nm wavelength intervals between 420 nm and 780 nm using a 10 nm bandwidth. A final fluorescence readout was produced by quantifying the images produced in ImageJ.HeLa, MDA-MB-231 and HepG2 cells were incubated with 5 μM of compound 22 in DMEM containing 10% serum for 60 min before washing and imaging in phenol red free DMEM by laser scanning confocal microscopy. Non-treated cells were also analysed as controls.5.6p ≥ 0.05) datasets allowing for parametric data assessment using the Dunnett's test. All Statistical analyses were conducted using GraphPad Prism 7.02 .Statistical analyses of parasitological assays were performed using a nonparametric two-way ANOVA followed by Least Significant Difference post-hoc correction . During genotoxicity studies, Bartlett and Shapiro-Wilk tests were used to confirm homogenous variance and normal distribution for p53 and ɣH2AX (S. mansoni screening and cell toxicity study), NE, LJL, CL (S. japonicum and S. haematobium screening), DSGH, REB (genotoxicity study), ES . Resources: JFT, HW . Manuscript preparation: GP, KFH. Manuscript revision: GP, NE, LJL, CL, DSGH, JFT, HW, MB, SF, ATJ, CRC, IC, AB, KFH.Conceived and designed the experiments: GP, AB, KFH. Performed the experiments: GP, MB, SF (compound synthesis and characterisation), GP  for financially supporting this project. The CDIPD team acknowledges the Bill and Melinda Gates Foundation (OPP1171488) for support. Hamsters and mice infected with S. haematobium and S. japonicum, respectively, were provided by the National Institute of Allergy and Infectious Diseases (NIAID) Schistosomiasis Resource Centre of the Biomedical Research Institute  through the National Institutes of Health (NIH)-NIAID Contract HHSN272201700014I for distribution through BEI Resources.KFH, GP and AB thank the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Method. We established a monolayer endothelial cell model by culturing human umbilical vein endothelial cells (HUVEC) in vitro, used tumor necrosis factor (TNFα) and HIF-1α inhibitor 2-methoxyestradiol (2ME2) to act on HUVEC, and at the same time constructed siRNA-transfected HUVEC to interfere with the expression of HIF-1α. The permeability of monolayer endothelial cells was measured by transwell chamber method, the concentration of MMP-9 in the supernatant was measured by ELISA method, the expression of key molecules related to permeability  was measured by RT-PCR and Western blot method, and the localization and expression of claudin-5 and ZO-1 were measured by immunofluorescence method. We searched for 7 HIF-1α hypoxia response elements within 4000 bp before the transcription start site in the MMP-9 promoter region, constructed the MMP-9 promoter-luciferase reporter gene recombinant plasmid, transfected and stimulated HUVEC with TNFα, and detected the effect of 7 hypoxia response element plasmids on the transcription activity of MMP-9 promoter.  This study mainly explored the mechanism of capillary leakage caused by hypoxia-inducible factor-1α, the permeability of monolayer endothelial cells increased, and the concentration of MMP-9 in the cell supernatant increased. 2ME2 and HIF-1α-siRNA transfection can improve the above situation (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can inhibit the high expression of HIF-1α and MMP-9 caused by TNFα, thereby increasing the expression of claudin-5 and ZO-1 (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can reduce the inhibition of TNFα on the expression of cell membrane protein claudin-5 and tight junction protein ZO-1. Element 1, element 5, and element 7 are the sites where HIF-1α interacts with MMP-9 at the transcription level.  Under the action of TNFα can increase the permeability of monolayer epithelial cells by inducing the high expression of MMP-9, leading to capillary leakage. Its target is at the −3798 bp, −1878 bp, and −1489 bp points of the transcription initiation site in the MMP-9 promoter region. This study shows that HIF-1 Vascular leakage is the main reaction of tissue damage . The desα subunit and β subunit. HIF-1α (hypoxia-inducible factor-1a) encodes the α subunit of the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 activates the transcription of many genes, including genes involved in energy metabolism, angiogenesis, and apoptosis and genes generating other protein products that can increase oxygen transfer or promote metabolism to adapt to hypoxia, as a main regulator in the homeostatic response to cells and the whole body hypoxia. HIF-1 plays an important role in embryonic angiogenesis, tumor angiogenesis, and pathophysiology of ischemic diseases. For example, it has been reported that HIF-1α promotes tumor chemotherapy resistance by recruiting GDF15-induced tumor-associated macrophages in colorectal cancer [α/NF-kappaB by competitively binding miR-33a-5p to inhibit the occurrence of infantile hemangioma [α [β-induced chondrocyte degeneration, FBW7 regulates the HIF-1α/VEGF pathway [α [α [HIF-1 is a heterodimer composed of l cancer ; silencimangioma ; under hgioma [α ; in the  pathway ; miR-210ay [α [α .Proteins of the matrix metalloproteinase (MMP) family are involved in the degradation of extracellular matrix in normal physiological processes, such as embryonic development, reproduction and tissue remodeling, and disease processes such as arthritis and metastasis. Most matrix metalloproteinases are secreted in the form of inactive proproteins and are activated when cleaved by extracellular proteases. MMP9 , as a member of the matrix metallopeptidase family, also plays an important role in many diseases and life processes. In dentistry, MP-8, MMP-9, and BOP can be used to assess the periodontal condition of orthodontic patients ; in otolα on vascular leakage. Therefore, this study explored the mechanism of HIF-1α inducing the high expression of MMP-9 and causing capillary leakage.However, there are still very few studies on the mechanism of HIF-1in vitro, using tumor necrosis factor (TNFα) and HIF-1α inhibitor 2-methoxyestradiol (2ME2) to act on HUVEC, and constructing siRNA-transfected HUVEC to interfere with the expression of HIF-1α. The permeability of monolayer endothelial cells was measured by transwell chamber method, the concentration of MMP-9 in the supernatant was measured by ELISA method, and the expression of key molecules  related to permeability was measured by RT-PCR and Western blot method. The localization of claudin-5 and ZO-1 was observed and the protein expression was reconfirmed by the immunofluorescence method. The MMP-9 promoter-luciferase reporter gene recombinant plasmid was constructed and transfected, TNFα was used to stimulate HUVEC, and the effect of 7 hypoxia response element plasmids on the transcription activity of MMP-9 promoter was detected.In this study, a monolayer endothelial cell model was established by culturing human umbilical vein endothelial cells (HUVEC) α group, adding DMEM high glucose medium containing 50 ng/ml TNFα; TNFα + 2ME2 (0.1 uM) group, adding DMEM high glucose medium containing 50 ng/ml TNFα and 0.1 uM 2ME2; TNFα + 2ME2 (0.01 uM) group, adding DMEM high glucose medium containing 50 ng/ml TNFα and 0.01 uM 2ME2; TNFα + 2ME2 (0.001 uM) group, adding DMEM high glucose medium containing 50 ng/ml TNFα and 0.001 uM 2ME2; HIF-1α siRNA-negative control group, negative HIF-1α siRNA was used to transfect HUVEC, and after monolayer cells were formed, 50 ng/ml TNFα was added to act for 24 hours; HIF-1α siRNA-positive transfection group, positive HIF-1α siRNA was used to transfect HUVEC, and after monolayer cells were formed, 50 ng/ml TNFα was added to act for 24 hours. This study was approved by the institutional ethical review board of the 960th Hospital of the PLA Joint Logistics Support Force, China.HUVEC cells were cultured in DMEM high glucose medium  and placed in an incubator , and the cell culture medium was replaced every 2 days until it grows and converges to 70–80% passage. 3–4 times passage was taken for experiment and divided into 7 groups, namely, the normal control group, only adding DMEM high glucose medium containing 10% fetal bovine serum; TNF5/well) to establish a complete monolayer endothelial cell model. After drug action as grouped, we washed with PBS for 3 times, seeded 100 ul PBS which contains FITC‐Dextran40  100 mg/L in the upper transwell chamber, filled the lower chamber with 600 ul PBS for 1hours, collected the PBS in the lower chamber, measured the intensity of FITC fluorescence with a fluorescence spectrometer, established a standard curve between fluorescence intensity and FD40 concentration, and used FD40 concentration to reflect the permeability of monolayer endothelial cells.HUVEC was seeded in the upper chamber of the transwell (1 × 105/well). After stimulation with drugs as grouped, we took the supernatant and tested the concentration of MMP‐9 by ELISA method. The kit was purchased from Abcam (ab246539) and used following the kit instructions. The absorbance was measured at 450 nm with an ultraviolet spectrophotometer.HUVEC was seeded into a 96-well plate  to detect the mRNA abundance by real‐time quantitative PCR. The PCR conditions are as follows: 95°C (30 s) 1 cycle, and 95°C (5 s), 60°C (34 s), 40 cycles. The primers are as follows: HIF-1After establishing a monolayer HUVEC model, we added drugs as grouped and extracted total protein of the cells for detection.μg, and electrophoresis was imposed at 100 V for 1 to 2 hours. The antibody was purchased from Abcam, USA. The primary antibodies were rabbit antibodies, anti-MMP-9 antibody (ab76003), with a dilution ratio of 1 : 1000, anti-HIF-1α antibody (ab51608), with a dilution ratio of 1 : 500, anti-claudin 5 antibody (ab131259), with a dilution ratio of 1 : 5000, and the anti-ZO-1 antibody (ab276131), with a dilution ratio of 1 : 1000. The secondary antibodies were goat anti-rabbit antibodies (ab6721), with a dilution ratio of 1 : 3000. ImageJ software was used to quantify the gray value of the resulting graph and drew a statistical graph.An equal volume of 2× Laemmli sample buffer was added to the supernatant, and the supernatant mixture was boiled at 100°C for 5 minutes and stored at −20°C. Then, the centrifuge tube containing the supernatant mixture was thawed at 37°C. The same amount of protein and maker was loaded into the SDS-PAGE gel wells, with a sample amount of 20–30 The immunofluorescence experiment was carried out according to the previously published method . HUVEC wE. coli Trans5α, and the plasmid was extracted for enzyme digestion identification. The identified target DNA fragments were connected to pGL3-Basic vector to construct a luciferase reporter plasmid, E. coli Trans5α was transformed, amplified, sequenced, and identified, the luciferase reporter plasmid already constructed was extracted, the plasmid was transfected into HUVEC, and the activity of the dual-luciferase reporter gene after TNFα stimulation was detected.By searching Ensemble and NCBI, two major gene databases, it was found that the human MMP-9 genome contains 7 hypoxia response elements 4000 bp before the transcription start site in the promoter region. According to the hypoxia response element position of the human MMP-9 promoter sequence, promoter fragments of different lengths were designed, and then the PCR primers of each promoter fragment were designed and synthesized. The whole blood DNA of healthy volunteers was extracted by the NaI method, human genomic DNA was used as a template, the promoter fragment was obtained by PCR amplification, the target DNA was cloned by T-A and transformed into t-tests to calculate P values. The result of P < 0.05 has statistical significance.All analyses involved in this study used unpaired α group was 2.339 ± 0.149 mg/l, which was significantly higher than that of the normal control group, which was 1.485 ± 0.137 mg/l. In the TNFα + 2ME2 group, after giving 0.1 uM, 0.01 uM, and 0.001 uM 2ME2 for coculture, the concentrations of FD40 were 1.927 ± 0.147, 1.803 ± 0.136, and 2.057 ± 0.193 mg/l, respectively. Compared with the TNFα group, P < 0.05, suggesting improved permeability  in the HIF-1α siRNA-negative control group, suggesting that after siRNA knocked down HIF-1α expression, the permeability decreased  under different treatment conditions, we tested the concentration of FD40 in different treatment groups. The concentration of FD40 in the lower chamber of the TNFeability . The conecreased .α group was 926.6 ± 322.6 pg/ml, which was significantly higher than that of the normal control group of 348.2 ± 130.1 pg/ml. In the TNFα + 2ME2 group, after giving 0.1 uM, 0.01 uM, and 0.001 uM 2ME2, respectively, for coculture, the concentrations of MMP9 were 655.9 ± 213.66, 696.9 ± 235.27, and 839.5 ± 284.28 pg/ml, respectively, which were lower than those in the TNFα group, P < 0.05 , indicating that after siRNA knocks down the expression of HIF-1α, the concentration of MMP9 in the supernatant is reduced ).The concentration of MMP9 in the supernatant of the HIF-1α mRNA in the normal control group was 1.006 ± 0.036, and the relative expression in the TNFα group was 2.080 ± 0.267, which was significantly higher than that in the normal control group, P < 0.01; in the TNF α + 2ME2 0.1 uM group, the TNFα + 2ME2 0.01 uM group, and the TNFα + 2ME2 0.001 uM group, the relative expression of HIF-1α mRNA was 0.292 ± 0.099, 1.464 ± 0.066, and 1.795 ± 0.172, respectively. 0.1 uM and 0.01 uM 2ME2 can inhibit the high expression of HIF-1α caused by TNFα, P < 0.01. The inhibitory effect disappeared with 0.001 uM 2ME2, P=0.145 . Compared with the TNFα group, the expression of MMP-9 protein in the TNFα + 2ME2 0.01 uM group and the TNFα + 2ME2 0.001 uM group was significantly reduced, and the expression of MMP-9 protein in the TNFα + 0.001 uM 2ME2 group was slightly increased, P=0.408 , suggesting that after siRNA knocked down the HIF-1α expression, the HIF-1α expression caused by TNFα interfered. The relative expression of MMP-9/β actin protein in the HIF-1α siRNA-positive transfection group was 0.278 ± 0.006, which was lower than 0.517 ± 0.021 in the HIF-1α siRNA-negative control group (P < 0.01), indicating that after siRNA interfered with the HIF-1α expression, the MMP-9 high expression was inhibited. The relative expression of claudin-5/β actin protein in the HIF-1α siRNA-positive transfection group was 0.498 ± 0.018, which was lower than 0.241 ± 0.011 in the HIF-1α siRNA-negative control group (P < 0.01), suggesting that after siRNA interfered with the HIF-1α expression, the expression of claudin-5 was promoted. The relative expression of ZO-1/β actin protein in the HIF-1α siRNA-positive transfection group was 0.304 ± 0.004, which was lower than 0.032 ± 0.004 in the HIF-1α siRNA-negative control group (P < 0.01), suggesting that after siRNA interfered with the HIF-1α expression, the expression of ZO-1 was promoted . 2ME2 and HIF-1α-siRNA transfection can improve the above situation (P < 0.05). TNFα is a proinflammatory factor secreted by macrophages and endothelial cells, which is related to the increase of plaque load [α can lead to the loss of adhesion protein, thereby increasing cell permeability [α increased significantly, indicating an increase in cell permeability, which is consistent with previous reports. However, with the addition of the HIF-1α inhibitor 2-methoxyestradiol (2ME2), the increasing trend of FD40 concentration was inhibited, and with the increase of the 2ME2 concentration, the inhibitory effect gradually increased. Besides, the addition of HIF-1α siRNA would also significantly decrease the concentration of FD40, suggesting that TNFα may affect cell permeability by regulating HIF-1α . 2ME2 and HIF-1α-siRNA transfection can inhibit the high expression of HIF-1α and MMP-9 caused by TNFα, thereby increasing the expression of claudin-5 and ZO-1 (P < 0.05). 2ME2 and HIF-1α-siRNA transfection can improve the inhibition of TNFα on the expression of cell membrane protein claudin-5 and tight junction protein ZO-1. Element 1 (−3798 bp), element 5 (−1878 bp), and element 7 (−1489 bp) were the sites where HIF-1α interacted with MMP-9 at the transcription level.All in all, this study showed that, under the action of TNF


S)-N-methyl-1-phenylethan-1-amnonium chloride (1), usually represent by-products in amination processes of chlorosilanes. Hirshfeld surface analysis and Hirshfeld atom refinement (HAR) were performed to obtain detailed information about the crystal packing and the exact position of the hydrogen atoms of compound 1.Ammonium chlorides, such as the title compound , can be synthesized starting from (S)-N-methyl-1-phenylethan-1-amine (2). Compound 2 upon addition of HCl·Et2O leads to crystallization of compound 1 as colorless blocks. The configuration of compound 1 is stable as well as preserved in space group P212121. Ammonium chlorides, like the title compound, are often observed as undesirable by-products in aminosilylation of chlorosilanes. Additionally, these by-products are usually soluble in selected organic solvents, which require difficult separation steps. Therefore, detailed studies on structural features and intermolecular interactions performed by Hirshfeld atom refinement (HAR) using NoSpherA2  and Hirshfeld surface analysis were used to address structural issues on that separation problem.The title compound C al. 2021. Chem. S The absolute configuration of the chiral ammonium chloride 1 in the measured crystal can be assigned with the (S)-configuration using the Cahn–Ingold–Prelog (CIP) prioritization .Compound sp3—N bond lengths are in a range of 1.4816 (4) Å (N1—C3) and 1.5034 (4) Å (N1—C3), which are typical for most structurally analyzed ammonium salts . All conformations were taken from the solid-state structure at the start of the optimization. The result of the calculation provides smaller Csp3—N bond lengths than from the solid-state structure in principle. The calculated bond lengths are 1.4762 Å (N1—C3) and 1.4946 Å (N1—C2).In the literature, known Cb⋯Cl1) was not described sufficiently with the addition of the used potential and basis set. Therefore, a large deviation can be observed from the analyzed distance compared to the crystal structure. Further analyses concerning supramolecular interactions are discussed in detail in the next section.Hydrogen-bond lengths as well as associated angles are shown in Table 1et al., 2000The stereogenic carbon center features a tetrahedral geometry, which is slightly distorted as shown by the angle of 107.44 (2)° (C1—C2—N1). However, the geometric distortion of a tetrahedral carbon center has been observed in many compounds with different substituents  , however represent the most intense contacts on the surface Fig. 4. HydrogeCrystalExplorer2021. As can be seen in Fig. 21 forms one-dimensional chains along the a-axis direction in the crystal structure. These can be attributed to the strong C—H⋯Cl interactions already mentioned, as well as additional C—H⋯π interactions, which are illustrated in Fig. 2In addition to the influence of C—H⋯Cl contacts, the influence of possible π-interactions was analyzed by 1. Depicted examples found in Cambridge Structural Database cyclohex-4-ene-1,2-diaminium dichloride monohydrate, C22H32N2O2Cl cyclopentan-1-aminium chloride, C16H24ClNO2 -(cyclohexan-(R)-2-ol)]-(R)-a-methylbenzyl ammonium chloride, C14H22NOCl ethyl][(1R)-1-phenylethyl]ammonium chloride, C19H22NCl -2-methoxy-1-(1-phenylethylamino)cyclopentanecarboxamide hydrochloride, C15H23N2O2Cl  hydrogen bonds. The smallest observed hydrogen bond of the two literature known compounds amounts to 2.2 Å. These longer distances could be due to the more sterically demanding substituents, which are less pronounced in compound 1. Moreover, in terms of the crystal packing, the compounds do not exhibit one-dimensional chains, as C—H⋯π contacts were not observed. This unique feature of (S)-N-methyl-1-phenylethan-1-aminium chloride (1), which does not appear in the literature compounds, could again be attributed to the steric crowding of the other compounds.There are some crystallographically characterized examples based on the structure of compound 1 is illustrated in the scheme below. (S)-N-methyl-1-phenylethan-1-amine (2) (1.48 mmol) was dissolved in diethyl ether (5 mL). A 2 M solution of HCl in diethyl ether (1.78 mmol) was added dropwise. The solution was stored at 298 K for three days. Afterwards all volatile compounds were removed and the raw product was washed with cold n-pentane (1 ml). (S)-N-methyl-1-phenylethan-1-aminium chloride (1) was isolated as colorless crystalline plates.The reaction scheme for the synthesis of compound 1H NMR : δ = 1.88 , 2.46 , 1.65–1.90 , 7.38–7.45 , 7.60 , 9.78 , 10.17  ppm.13C NMR : δ = 20.5 , 31.0 , 60.2 , 127.9 , 129.5 , 135.6  ppm.{1H}CHN analysis: calculated: C = 62.97%, H = 8.22% N = 8.16%; Found: C = 62.6%, H = 7.9%, N = 7.8%.Rf:  = 0.20.b were refined freely using independent values of each Uiso(H). Hirshfeld atom refinements  global, I. DOI: Click here for additional data file.10.1107/S2056989021013645/dx2041Isup2.cmlSupporting information file. DOI: 2132333CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


According to data estimated by the WHO, primary liver cancer is currently the fourth most common malignant tumor and the second leading cause of death around the world. Hepatocellular carcinoma (HCC) is one of the most common primary liver malignancies, so effective therapy is highly desired for HCC.l-Aspartic acid)-poly(ethylene glycol)/combretastatin A4 (CA4-NPs) was aimed to significantly disrupt new blood vessels in tumor tissues for targeted hepatic tumor therapy. Here, PEG-b-PAsp-g-CA4 showed significantly prolonged retention in plasma and tumor tissue. Most importantly, CA4-NPs were mainly distributed at the tumor site because of the triple target effects—enhanced permeability and retention (EPR) effect, acid-sensitive (pH = 5.5) effect to the tumor microenvironment (TME), and good selectivity of CA4 for central tumor blood vessel. Considering that CA4-NPs might induce severe hypoxic conditions resulting in high expression of HIF-1α in tumor tissues, which could induce the overexpression of PD-L1, herein we also used a programmed death-ligand 1 antibody (aPD-L1) to prevent immunosuppression. This way of complementary combination is able to achieve an ideal treatment effect in tumor site where CA4-NPs and aPD-L1 could respond to the inner area and peripheral area, respectively. As a result, a significant decrease in tumor volume and weight was observed in the combination group of CA4-NPs plus aPD-L1 compared with CA4-NPs or aPD-L1 monotherapy in subcutaneous Hepa1-6 hepatic tumor models.In this study, the use of poly(We presented a new idea that co-administration of CA4-NPs and aPD-L1 possessed notable anti-tumor efficacy for HCC treatment.The online version contains supplementary material available at 10.1186/s12951-021-00865-w. Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide and more than half of the new cases and deaths occurred in Asia , 2. As il-Aspartic)-polymeric-drug conjugates have consistently been used for drug development as PAsp is biocompatible, biodegradable, and easily modified to be a drug conjugate , vortexed for 5 min, and centrifuged at 10,000 rpm for 10 min. The supernatant was filtered by using 0.22 μm filters before HPLC measurement. The pharmacokinetics assay of PEG-b-PAsp-g-CA4 and CA4P was performed by fluorescence detector HPLC (FLD-HPLC) with a fluorescence detector at 290 nm Ex and 380 nm Em, and the elution was performed with methanol and water (v/ v = 7: 3) pumped at a flow velocity of 1.0 mL min−1 at 40 °C.Pharmacokinetics was characterized by measuring CA4 levels by collecting the eye arterial blood of SD female rats (about 250 g). Specifically, six rats were divided into two groups of 3 each, and they were injected with free CA4P (50.0 mg kg7 cells) in the right flank. When tumor reached 150–200 mm3, mice were randomly divided into two groups (free Cy5.5 and PEG-b-PAsp-g-CA4-Cy5.5) at an identical 50 μg kg−1 Cy5.5. 2,6, 12 and 24 h after injection, NIR fluorescent images were captured by in vivo imaging system . Finally, mice were sacrificed and heart, liver, spleen, lung, kidneys and tumor of each mouse were harvested for more detailed comparison.To explore the tumor-targeting capability of CA4-NPs in vivo, Hepa1-6-bearing mice were i.v. injected with Cy5.5-loaded nano drugs followed by near-infrared optical imaging. Briefly, mice were subcutaneously inoculated with Hepa1-6 cells (1 × 107 cells) in the right flank. When tumor reached 150–200 mm3, mice were randomly divided into two groups (free CA4P and PEG-b-PAsp-g-CA4) via the tail vein at dose of 40 mg CA4/kg. At 24 h, the mice were killed and their tumors, hearts, livers, spleens, lungs, kidneys were collected. Tissue samples were washed in ice-cold saline blotted with paper towels to remove excess fluid and weighed. Then the tumors and main organs were homogenized in water (1 g tissue per 1 mL) to get suspensions. And 100 μL tissue samples were deproteinized into 300 μL of acetonitrile by vortex for 5 min, and centrifuged at 10,000 rpm for 10 min. The test conditions of tissue biodistribution study was performed the same as pharmacokinetics assay.To explore the tissue biodistribution study further, mice were subcutaneously inoculated with Hepa1-6 cells  for antigen retrieval. After treating in blocking medium (5% BSA PBS buffer) for 20 min, the tumor tissue slices were first incubated with a monoclonal primary antibody , and incubated with a fluorescently labeled secondary antibody (Cy3/AF488/AF594), then imaged by confocal laser scanning microscopy (CLSM).7 cells mL−1, 200 µL) and subcutaneously injected near the mammary region of mice.Healthy female C57BL/6 mice (20 ± 2 g) were purchased from Charles River (Beijing), and handling procedures were according to the guidelines of the Regional Ethics Committee for Animal Experiments of China Pharmaceutical University. Hepa1-6 tumor-bearing mice were selected as the animal model to assess the anti-tumor effect. Hepa1-6 cells were harvested after cell culture. Then, cells were suspended in PBS , CA4-NPs (20 mg kg−1) (group 2), CA4-NPs (40 mg kg−1) (group 3), aPD-L1 (group 4), CA4-NPs (20 mg kg−1) + aPD-L1 (group 5), and CA4-NPs (40 mg kg−1) + aPD-L1(group 6). The ratio of CA4 amount and body weight was about 5 mg kg−1 or 10 mg kg−1. The dosage of aPD-L1 was 50 µL for each mouse. The tumor size and body weight were recorded every day during the treatment. The mice were sacrificed after 15 days, and then the tumors and major organs were isolated and stained by hematoxylin & eosin (H&E). Besides, Ki67 and TUNEL immunostaining were also applied for all the tumors. Finally, the survival time of the remaining mice for each group were monitored additionally until day 60 and the survival curves were produced.When the tumor volume reached about 100 mmTo evaluate the level of IFN-γ and IL-4, frozen tumors were homogenized in PBS solution at 4 °C. All homogenized samples were centrifuged and the supernatants were stored at − 80 °C before analysis. Meanwhile, the blood samples were centrifuged to obtain the serum. Then cytokines both in mice serum and tumor were analyzed with corresponding ELISA kits  according to the manufacturers’ instruction.Data were calculated and processed as mean ± SD. Comparison between groups was analyzed with Student’s t-test or one-way analysis of variance (ANOVA). Statistical differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.Additional file 1:Figure S1. Images of the excised tumors after different treatments; shown are PBS-treated mice and mice receiving after injection with CA4-NPs (20 mg kg-1), CA4-NPs (40 mg kg-1), aPD-L1, CA4-NPs (20 mg kg-1) + aPD-L1, CA4-NPs (40 mg kg-1) + aPD-L1, CA4P (40 mg kg-1), n=10. Figure S2 Tissue distributions of CA4 in mice receiving free CA4P and PEG-b-PAsp-g-CA4 at 24 h. Figure S31H NMR spectra of BLA-NCA.