While PXT (II) in an open V-shaped conformation that has the B-ring shallowly inserted in the β-CD cavity, PXT (I) in a closed U-shaped structure is mostly entirely embedded in the β-CD dimeric cavity, of which the A-ring is deeply inserted in the main β-CD cavity. However, PXT molecules in both crystal forms are similarly maintained in the CD cavity via host–guest N–H···O5/O6 H-bonds and C/O–H···π(B/C) interactions and β-CDs have similar 3D arrangements, channel (II) vs. screw-channel (I). Further theoretical explorations on the β-CD–PXT thermodynamic stabilities and the PXT conformational stabilities based on their potential energy surfaces (PESs) have been completed by DFT calculations. The 2:1 β-CD–PXT complex with the greater presence of dispersion interactions is more energetically favorable than the unimolar complex. Conversely, whereas free PXT, PXT (II) and PXT in complex with serotonin transporter are more energetically stable, PXT (I) is least stable and stabilized in the β-CD cavity. As SSRIs could lessen the COVID-19 severity, the CD inclusion complexation not only helps to improve the drug bioavailability, but also promotes the use of antidepressants and COVID-19 medicines concurrently.Depression, a global mental health problem, is prevalent during the coronavirus disease 2019 (COVID-19) pandemic and can be efficiently treated by selective serotonin reuptake inhibitors (SSRIs). Our study series aims at forwarding insights on the β-cyclodextrin (β-CD)–SSRI inclusion complexes by X-ray crystallography combined with density functional theory (DFT) calculation. Here, we report a new crystal form (II) of the 1:1 β-CD–paroxetine (PXT) complex, which is inspired by the reported 2:1 β-CD–PXT complex , reflecting an elusive phenomenon of the polymorphism in CD inclusion complexes. The β-CD–PXT polymorphism stems from the PXT conformational flexibility, which is defined by torsion angles  Depression is a common mental illness worldwide, affecting 3.8% of the population or ~280 million people, and 5.0% of adults suffer from it . The emeκ, ε in Depression can be efficiently treated by selective serotonin reuptake inhibitors (SSRIs)—second generation—and tricyclic antidepressants (TCAs)—first generation medications. The antidepressants approved by the US Food and Drug Administration  include,N-oxide , ψ [C1(n + 1)–O4(n)–C4(n)–C5(n)], of which their sum of averages is close to null for a round CD structure , ω [O5–C5–C6–O6] describe the freely rotating O6–H groups, of which their values are largely varied and hence excluded from the calculation of rms fits.The CD structure comprises six parameters that can be categorized into three groups . Group 1tructure ; (iii) tO4 atoms . Group 22O  and ε [C3–C7–O8–C9] in various lattice circumstances, of which their crystal data can be retrieved from two large crystallographic databases, including the Cambridge Crystallographic Data Center  of the relevant X-ray structures after rounding for PES scans are classified into three groups as follows (see × and ○ in κ and varied ε (30–310°), and (ii) the complete-geometry optimization of the five structures with the minimum total energy from the PES scans to obtain the conformer at global minimum energy.The PXT structural flexibility plays a pivotal role in governing its pharmacological functions. The distinct conformations of PXT arising from two torsion angles am.ac.uk  accessedrcsb.org  accessedEtot) of five PXT conformers with fixed κ = 90°, 170°, 270°, 300° and 320° and scan coordinate ε = 30–310° with an increment of 10°. After the PES rigid scans, to obtain a minimum total energy (Etot_min) for each conformer  = , while those of the other four PXT conformers were at  for PXT (I),  for 5I6X,  for 6AWN and  for 6VRH. Clearly, all five PESs have a common feature of one saddle and two wells with the total energy differences and energy barriers of <1 and ~3 kcal mol−1 for PXT (II) partially and PXT (I) totally embedded in the β-CD cavity, and of ~4 and ~4 kcal mol−1 for PXT in complex with SERT , see ○ in , PXT (IIEtot_min from the PES scans including PXT–β-CD form II (ε = 270°) and the rest (ε = 80°–180°) are subsequently used for full-geometry optimization. The energy minimization provided genuine global minimums of the completely optimized structures, which are lower than Etot_min of the structures from constrained PESs. The corresponding κ, ε, Eopt are 179.8°, 276.6°, −1116.33014 Hartree ; 80.4°, 182.0°, −1116.32861 Hartree (PXT–2β-CD form I); and 299.6°, 178.7°, −1116.33244 Hartree .The PXT structures with κ, ε) =  is the most stable and is 3.16 kcal mol−1 more stable than PXT (I), which is the least stable with rather high Etot_min at  =   coordinates of the X-ray, PES scanned and fully optimized structures  a,b. Thisructures a,c. (iiiructures c. After function .PXT HCl hemihydrate (≥98%) was obtained from Acros Chemicals (code 462630). β-CD (≥95%) was purchased from Cyclolab, Budapest, Hungary (code CY-2001). Absolute EtOH (≥99.8%) was supplied by Liquor Distillery Organization, Excise Department, Chachoengsao, Thailand. All chemicals were used as received. The ultrapure water was obtained from a Milli-Q Water System.P21 (form I), which were prepared by kneading of the solid mixture, dissolving in pure water at 313 K and solvent evaporation at 293 K [Several attempts were made to reproduce colorless, prismatic single crystals of the 2:1 β-CD–PXT base inclusion complex in the monoclinic, space group at 293 K . Here, wat 293 K  and the at 293 K . Note that 293 K . Moreoveat 293 K  and β-CDat 293 K  yielded at 293 K .2O 17 mg (0.022 mmol) were dissolved in 750 mL of 50% (v/v) EtOH–H2O at 323 K, giving a concentrated complex solution. A vial containing the complex solution was left to stand still in an air-conditioned lab room at 298 K. After a month of slow solvent evaporation (during the work from home period due to the 2nd wave of the COVID-19 pandemic in Bangkok in January 2021), it was ultimately fruitful to obtain colorless, thick plate-like crystals. Surprisingly, preliminary X-ray analysis revealed a new crystal form, i.e., form II of the 1:1 β-CD–PXT HCl inclusion complex in the triclinic space group P1.β-CD 50 mg (0.044 mmol) and PXT HCl·0.5HRint values were 67,271/23,459/14,844, 99.8% and 0.0296, respectively.Different pieces of thick plate-like single crystals were selected, cut and separately mounted in a thin-walled glass capillary . They were screened for consistent unit cell constants and sufficient diffracting power. All crystals belonged to the triclinic system with similar unit cell parameters, suggesting a new crystal form of the β-CD–PXT complex. A cut rod-shaped crystal (0.32 × 0.34 × 0.44 mm) with the best diffracting power was chosen for X-ray data collection at 296 K to 0.70-Å atomic resolution on a Bruker APEXII CCD area-detector diffractometer . Data processing was carried out according to standard procedures using the APEX2 software suite : (i) intThe crystal structure of the β-CD–PXT complex in the triclinic system (form II) was solved by the intrinsic phasing method with SHELXTL XT , providi2–O6–H groups  in β-CD and 5 of 7 water molecules distributed over 12 positions (site occupancy factors (SOFs) = 0.3–0.6) in the intermolecular interstices; 2 water molecules were fully occupied. Plus, the peak cluster was better modeled as the disordered ethanol site (SOF = 0.4). All non-H atoms were refined anisotropically by full-matrix least-squares on F2 using SHELXTL XLMP [Uiso = 1.2Ueq(C)(aromatic); C−H = 0.98 Å, Uiso = 1.2Ueq(C)(methine); C−H = 0.97 Å, Uiso = 1.2Ueq(C)(methylene); C−H = 0.96 Å, Uiso = 1.5Ueq(C)(methyl); and N−H = 0.89 Å, Uiso = 1.2Ueq(2° ammonium). The H-atoms of β-CD OH groups and two fully occupied water molecules, O1W and O2W, were initially located by difference Fourier maps; those of disordered water sites could not be found. Then, the OH H-atoms were refined using ‘AFIX 147’ or ‘AFIX 83’ with restraints O−H = 0.84 Å, Uiso = 1.5Ueq(O). The water H-atoms were refined with DFIX restraints to idealized geometry (O−H 0.96 Å and H∙∙∙H 1.52 Å) and with ‘AFIX 30’ constraint Uiso = 1.5Ueq(water). Moreover, to prevent short H∙∙∙H distances in the refinement, BUMP antibumping restraints were applied. The refinement converged to a final R1 = 0.0633. For more details of data collection and refinement statistics, see The disorders of β-CD O6–H groups and solvents caused trouble in the refinement. There were 2–3 doubly disordered C6–HXTL XLMP , except R1 = 0.0662 deserved further notes for comparison [The crystal structure refinement of the 2:1 β-CD–PXT base (form I) with a final mparison . Due to 2–O group bridge the A- and C–D-rings of PXT. This is to attain thermodynamically favorable CD inclusion complexes with optimum host–guest interactions, reflecting the importance of the induced-fit mechanism [Both host CD and guest drug exhibit conformational flexibility to an extent, particularly in this case, where the primary hydroxyls O6–H of β-CD and the CHechanism . Hence, echanism , olive pechanism , coffee echanism  for the echanism , SSRIs [echanism  for the 2O , indicating the polymorphism in the β-CD encapsulation of PXT. To evaluate the host–guest interactions in the two crystal forms, we considered the 2:1 and 1:1 β-CD–PXT base for the respective forms I and II without water of hydration. The corresponding atomic coordinates of both phases from X-ray analysis, of which the underestimated X–H bond distances were corrected to neutron hydrogen distances: C–H, 1.083 Å; N–H, 1.009 Å; and O–H, 0.983 Å [Estb and ΔEint) of the 2:1 and 1:1 host–guest complexes were calculated using Equations (1) and (2).Ecpx, Emβ-CD_opt and ED_opt are the molecular energies from the full-geometry optimization of complex, monomeric (m = 1)/dimeric (m = 2) host β-CD and drug PXT, respectively; Emβ-CD_sp and ED_sp are the corresponding single-point energies in the complexed states.Thus far, there have been two crystal modifications of 2β-CD·PXT base·28H1—form I ) and β-C 0.983 Å . The nor 0.983 Å  on a DELEints with the dispersion-correction (B97D) and the BSSE correction of the structures optimized at the B3LYP/6-31+G*/4-31G level.In the vicinity of weak intermolecular interactions, such as CH···π type, which primarily stabilize β-CD inclusion complexes with TCAs ,33 and S2–O- group on C3. The B-ring had a more or less perpendicular torsion angle with respect to the plane passing through C2–C3–C5–C6 of the A-ring, as found in the β-CD–PXT form II, which was used for the PES rigid scans.A thorough conformational comparison in our previous work  and the ε and κ, respectively –(v) were fine-tuned for exact torsion angles κ, ε ; 87.2°,e TUZFIF ); 167.7°e TUZFIF ); −55.7°e TUZFIF ); and −3e TUZFIF ). To persee ○ in : (i) 90°Our study series aims at forwarding insights on the β-cyclodextrin (β-CD)–selective serotonin reuptake inhibitor (SSRI) inclusion complexes by X-ray crystallography combined with DFT calculation. This work focuses on the polymorphism of β-CD–paroxetine (PXT) complex and the distinctive nature of PXT conformational flexibility, which are inspired by the reported 2:1 β-CD–PXT complex .2O belongs to the triclinic, space group P1, whereas crystal form I of the 2β-CD·PXT base·28H2O pertains to the monoclinic, space group P21 [κ, ε around the bridge -CH2–O- connecting the A- and C–D-rings. While PXT (II) in an open V-shaped conformation has the B-ring shallowly inserted into the β-CD cavity, PXT (I) in a closed U-shaped structure is mostly entirely embedded in the β-CD dimeric cavity; the A-ring is deeply inserted in the main β-CD cavity. Even though the distinct PXT inclusion scenarios and conformations are notable, PXT molecules in both crystal forms are similarly maintained in the CD cavity via host–guest N–H···O5/O6 H-bonds and C/O–H···π(B/C) interactions and β-CDs have similar 3D arrangements, channel (II) vs. screw-channel (I).Here, we add the 1:1 β-CD–PXT complex to the list of elusive polymorphisms in CD inclusion complexes. Crystal form II of β-CD·PXT HCl·0.4EtOH·7Hroup P21 . The β-CEints) with corrections of the dispersion and the basis set superposition error (BSSE) indicate that the 2:1 β-CD–PXT complex with the greater presence of dispersion interactions is more energetically favorable than the unimolar complex. Conversely, PXT molecules existing in varied lattice environments, including the uncomplexed PXT HBr salt, PXT embedded in the β-CD cavity (forms I and II) and PXT in action while bound at the central site of the serotonin transporter (SERT) protein, suggest the contrary. Whereas free PXT, PXT (II) and PXT–SERT complex are more energetically stable, PXT (I) is the least stable. Here, the PXT conformational flexibility and stabilities are comprehensively investigated for the first time, suggesting the true nature of this pivotal SSRI drug. The coronavirus disease 2019 (COVID-19) pandemic caused havoc on both economics and physical and mental health. It seemed not to end by mass vaccination. As there is a growing body of evidence that SSRIs could lessen the COVID-19 severity [Further theoretical explorations on the β-CD–PXT thermodynamic stabilities and the PXT conformational stabilities based on their potential energy surfaces (PESs) have been completed by DFT calculations. The complex interaction energies (Δseverity ,7,8,9,10severity .


Surgically induced nerve damage is a common but debilitating side effect in oncological surgery. With the aim to use fluorescence guidance to enable nerve-sparing interventions in future surgery, a fluorescent tracer was developed that specifically targets myelin protein zero (P0).Cy5-P0101–125 was selected after initial solubility, (photo)physical and in vitro evaluation (including P0-blocking experiments). Cy5-P0101–125 (KD = 105 ± 17 nM) allowed in vitro and ex vivo P0-related staining. Furthermore, Cy5-P0101–125  enabled in vivo fluorescence imaging of the Sciatic nerve in mice after local intravenous (i.v.) administration and showed compatibility with a clinical fluorescence laparoscope during evaluation in a porcine model undergoing robot-assisted surgery. Biodistribution data revealed that i.v. administered [111In]In-DTPA-P0101–125 does not enter the central nervous system (CNS).Truncated homotypic P0 protein-based peptide sequences were C-terminally functionalized with the far-red cyanine dye Cy5. The lead compound P0101–125 has proven to be a potent nerve-specific agent that is able to target P0/myelin under in vitro, ex vivo, and in vivo conditions without posing a threat for CNS-related toxicity.The online version contains supplementary material available at 10.1186/s13550-021-00792-9. Surgery, and science therein, has come a long way since Mr. Gunning and Lord Thurlow, respectively, stated to each other in 1796: “there is no more science in surgery”, in reply “than there is in butchery” , 4, 6. INerve-specific fluorescence imaging has been poised as a means to allow high-resolution nerve identification in real time . For thiBuilding on the truncation of the extracellular portion of P0 Fig. B that wa2O/MeCN 95:5 to 0.1% TFA in H2O/MeCN 5:95 in 20 min (1 mL/min). Low-resolution mass spectrometry (LRMS) was performed on a Bruker Microflex LT/SH MALDI-TOF mass spectrometer using linear mode. High-resolution mass spectrometry (HRMS) was performed on a Waters Acquity H-class UPLC  using a Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column with a gradient of 0.1% FA in H2O/CH3CN 98:2 to 0.1% FA in H2O/CH3CN 60:40 in 1.8 min (0.6 mL/min) coupled to a high-resolution XEVO G2S-XTOF Mass Spectrometer . 1H and 13C NMR were performed on a Bruker Ascend 850 (850 MHz) equipped with a CryoProbe  in deuterated solvents. Crude peptides were analysed by a Waters Acquity UPLC-MS system using a Waters BEH C18 1.7 µm, 2.1 × 100 mm column, applying gradient from 5% CH3CN in H2O + 0.2% TFA to 75% CH3CN in 7 min.All chemicals were obtained from commercial sources and used without further purification. DMF was dried over 4 Å molecular sieves. High-pressure liquid chromatography (HPLC) was performed on a Waters HPLC system  using a 1525EF pump and a 2489 UV detector. For preparative HPLC, a Maisch ReproSil-Pur 120 C18-AQ 10 μM (250 mm × 20 mm) column  was used at a flow rate of 12 mL/min. For analytical HPLC, a Maisch ReproSil-Pur C18-AQ 5 μM (250 mm × 4.6 mm) column was used with a gradient of 0.1% trifluoroacetic acid (TFA) in HP01–25, P021–45, P041–65, P061–85, P081–105, P095–120 and P0101–125 , P041–65 (41% isolated yield), P061–85 (22% isolated yield), and P081–105 (85% isolated yield) could be synthesized in fair yields. P095–120 and P0101–125 could only be effectively synthesized using pseudoprolines In-DTPA-P0101–125) is described in Additional file Synthesis of labelled control compounds ide NP41 , and DTPCy5-P0101–125 was selected as lead compound and subjected to more detailed chemical analysis. Methods and results for assessment of the (photo)physical properties of Cy5-P0101–125  are described in Additional file 2.P0-expressing RT4 D6P2T myelinating Schwannoma cells  and nonIn line with their use by Whitney et al. , transgeD) of Cy5-P01–25, Cy5-P041–65, Cy5-P061–85, Cy5-P081–105, Cy5-P095–120 and Cy5-P0101–125 for P0 was performed using P0-expressing RT4 D6P2T cells and a previously described flow cytometric method In-DTPA-P0101–125, 10 MBq of the labelled tracer was injected intravenously (tail vein). The percentage of the injected activity per gram of tissue (%IA/g) was assessed at 2 h post-injection as previously described In-DTPA-P0101–125 helped rule out accumulation of P0101–125 (MW = 3024 Da) in the CNS. Obviously, the chance of toxic side effects is also impacted by dosing and uptake in non-target organs. In nuclear medicine, disease-specific tracers are therefore applied using a micro-dosing regimen (< 100 µg/patient). Although the ability of using fluorescence at micro-dosing levels looks promising [In line with P0 expression, biodistribution studies performed with the radiolabelled analogue romising , this toromising , and manromising , 53–55. romising . Recent 5 MW = 30 Da in thromising . Substanromising  Fig. 4 AWhile most image-guided surgery studies promote the use of near-infrared (NIR) Cy7 analogues , 54, 56,Although intraneural injection provides a perfect proof of principle in both mice and pigs Figs. A, still By truncating the P0 protein, we have been able to successfully create a nerve-specific fluorescent tracer that is able to specifically stain P0/myelin expression in the PNS.Additional file 1. Errors regarding SI table/scheme references were corrected.


Their structures were established on the basis of extensive spectroscopic analysis and X-ray crystallographic experiments. The new compounds 1 − 4 were assessed for their activities to induce lysosomal biogenesis through LysoTracker Red staining. Compound 2 significantly induced lysosomal biogenesis. In addition, compound 2 could increase the number of LC3 dots, indicating that it could activate the lysosomal-autophagy pathway.Euphopepluanones F − K (The online version contains supplementary material available at 10.1007/s13659-021-00301-4. Euphorbia are used in traditional medicines for treatments of digestive system disorders, skin ailments, infections, inflammation, and injuries world-wide + ion at m/z 693.2512 , corresponding to the molecular formular C35H42O13. The mass spectrum indicate compound 3 was 42 mass units more than compound 1, suggesting that one of hydroxyl group in 1 was acetylated in compound 3. On comparing its NMR data with those of 1 , and the absence of the OH-2 signal. Thus, the OH-2 in 1 was inferred as being replaced by an acetoxy group in 3, which was supported by the HMBC correlations from H-2 to the carbonyl carbon. Therefore, the structure of 3 was delineated as shown.The positive HRESIMS data of euphopepluanone J (4) possessed a molecular formula of C27H34O8 as deduced from its positive HRESIMS . The spectra data of 4 closely related to those of 5, except for the absence of acetate signals at C-5, C-7, and C-8, suggesting the replacement of them with hydroxyl groups. Indeed, the resonances of C-4, C-6 and C-9 were up-shielded , and C-5 and C-8 were down-shielded  in 4, further supporting the presence of hydroxy groups at C-5, C-7, and C-8, instead of acetoxy groups in compound 5. The relative configuration of 4 was assigned as that of 1 by the nearly identical ROSEY data of these two compounds. Furthermore, the similar electronic circular dichroism (ECD) spectra of compounds 1, 2, 3 and 4  D25 + 64.9 ; UV (MeOH) λmax (log ε) 195 (4.52), 229 (3.96) nm; IR (KBr) vmax 3481, 2977, 1743, 1723, 1650, 1454, 1374, 1277, 1224 cm −1; 1H and 13C NMR data, see Table m/z [M + Na]+ 651.2426 .Euphopepluanone F : a color2): a white amorphous powder; [α]D25 + 41.2 ; UV (MeOH) λmax (log ε) 195 (4.48), 226 (4.05) nm; IR (KBr) vmax 3436, 2975, 2934, 1748, 1726, 1644, 1453, 1374, 1273 cm −1; 1H and 13C NMR data, see Table m/z [M + Na]+ 691.2716 .Euphopepluanone I (3): a white amorphous powder; [α]D25 + 16.3 ; UV (MeOH) λmax (log ε) 195 (4.54), 229 (3.91) nm; IR (KBr) vmax 3447, 2981, 1747, 1728, 1633, 1453, 1372, 1239 cm −1; 1H and 13C NMR data, see Table m/z [M + Na]+ 693.2512 .Euphopepluanone J (4): a white amorphous powder; [α]D25 + 59.3 ; UV (MeOH) λmax (log ε) 195 (4.45), 226 (3.92) nm; IR (KBr) vmax 3436, 2970, 2933, 1710, 1631, 1452, 1380, 1278 cm −1; 1H and 13C NMR data, see Table m/z [M + Na]+ 509.2151 .Euphopepluanone K  Å, b = 17.0114(4) Å, c = 10.2133(3) Å, α = 90°, β = 114.3100(10)°, γ = 90°, V = 1583.54(8) Å3, T = 100(2) K, space group P21, Z = 2, μ(CuKα) = 0.839 mm−1, 17,488 reflections measured, 5524 independent reflections (Rint = 0.0390). The final R1 values were 0.0330 (I > 2σ(I)). The final wR(F2) values were 0.0852 (I > 2σ(I)). The final R1 values were 0.0330 . The final wR(F2) values were 0.0853 . The goodness of fit on F2 was 1.065. Flack parameter = 0.04(4). These data can be obtained free of charge from The Cambride Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.Crystallographic Data for Compound 1–4 was evaluated using HeLa cell line, which was cultured at 37 °C with 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (HyClone), 100,000 U/mL penicillin and 100 mg/mL streptomycin. HeLa cell was purchased from ATCC.The activity to enhance lysosomal biogenesis of compounds Briefly, HeLa cells with 85% cell density in 96-well plates were treated with individual compounds at 20 μM in triplicate. Three hours later, cells were grown in fresh medium containing LysoTracker Red DND-99 (0.2 μM) for 30 min. Then, medium was changed to LysoTracker-free medium and images were taken with ArrayScan Infinity . Positive compounds were subjected to validation by treating HeLa cells with different concentrations  and at 1, 3 and 6 h in triplicate and staining with LysoTracker Red DND-99.CFP-LC3 expressing HeLa cells were treated with indicated compounds and images were collected by confocal microscopy. For live-cell imaging, cells grown on glass-bottom dishes were observed directly. All samples were examined with an inverted Olympus FV1000 confocal microscope. Images were analyzed with FV10-ASW 4.0a Viewer.µL reaction mixture. A real-time PCR system  was used to quantify gene expression in triplicate. Amplification of the sequence of interest was normalized with the reference endogenous gene actin.Total RNA was isolated from HeLa cells by using TRIzol Reagent (Invitrogen) according to the manufacturer’s recommendation. A reverse-transcription kit (Promega) was used to reverse transcribe RNA (1 µg) in a 20 t tests were employed for statistical analyses with a level of significance of p < 0.05.Data analyses were carried out using Prism 5, and Student’s Supplementary file1 (DOCX 76991 kb)Below is the link to the electronic supplementary material.


To inquiry about mechanism of miR-100-5p/CDC25A axis in breast carcinoma (BC), thus offering a new direction for BC targeted treatment.In vitro experiments were used for assessment of cell functions.qRT-PCR was employed to explore miR-100-5p and CDC25A mRNA levels. Western blot was employed for detecting protein expression of CDC25A. Targeting relationship of miR-100-5p and CDC25A was verified by dual-luciferase assay. P < 0.05) while CDC25A was highly expressed. Besides, miR-100-5p downregulated CDC25A level. miR-100-5p had a marked influence on the prognosis of patients. The forced miR-100-5p expression hindered BC cell proliferation, migration and invasion, and facilitated cell apoptosis. Upregulated miR-100-5p weakened promotion of CDC25A on BC cell growth.In BC tissue and cells, miR-100-5p was significantly lowly expressed (Together, these findings unveiled that CDC25A may be a key target of miR-100-5p that mediated progression of BC cells. Hence, miR-100-5p overexpression or CDC25A suppression may contribute to BC diagnosis. Breast carcinoma (BC) accounts for 7–10% of all solid malignancies . At presvia targeting Interleukin-8 (IL-8) to hamper BC cell malignant progression (via targeting specificity protein 1 (SP1), indicating that miR-539 is a possible target for diagnosis of BC (MicroRNAs (miRNAs) are evolutionarily conserved . miRNA egression . miR-485gression . miR-539is of BC . Taken tis of BC . miR-100is of BC , glioblais of BC , gastricis of BC  and renais of BC . Nonethevia targeting Sirtuin6 (SIRT6) in colorectal cancer  database. Expression analysis and survival analysis were carried out on the target miRNA according to the obtained data. Differential mRNAs (DEmRNAs) were got through differential analysis by using “edgeR” package (—logFC—>2.0 and padj<0.01). Three databases miRDB, starBase, and mirDIP were used to predict target mRNAs of miR-100-5p. Candidate mRNAs were got by overlapping up-regulated DEmRNAs and predicted mRNAs of miR-100-5p, among which a mRNA having the highest correlation coefficient was picked as the objective of study.Mature miRNA  and mRNA  expression data of TCGA-BRCA dataset were obtained from TCGA , human BC cell lines T47D (BNCC339607), MDA-MB-231 (BNCC339911), MCF-7 (BNCC100137) and BT-474 (BNCC101989) were bought from BeNa Culture Collection  and cultured at 37 °C with 5% COMDA-MB-231, MCF-7, T47D cell lines: DMEM with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin;MCF-10A, BT-474 cell lines: RPMI-1640 medium with 10% FBS.miR-100-5p mimic (miR-mimic), mimic NC (miR-NC), pcDNA3.1 (oe-NC), and pcDNA3.1-CDC25A plasmid (oe-CDC25A) were acquired from GenePharma Company . Cell transfection recommended lipofectamine 2000 .® 7500 Real-Time PCR Systems  with miScript SYBR Green PCR Kit  and SYBR® Premix Ex Taq TM II . PCR conditions were as follows: 95 °C 10 s, 60 °C 20 s, 72 °C 20 s, 45 cycles. 2−ΔΔCt method was utilized for data analyzing. GAPDH and U6 were applied as endogenous controls. Primer sequences were exhibited in Total RNA was extracted from cells by TRIzol kit  according to instructions. Concentration was assayed using NanoDrop 2000 system . cDNA was generated by reversely transcribing miRNA with miScript IIRT kit  and mRNA with PrimeScript RT Master Mix  according to protocols. qRT-PCR was completed on Applied Biosystems3 cells/well). Ten µL CCK-8 solution  was added at 24, 48, 72 and 96 h for 2 h of incubation under standard conditions. Absorbance value at 450 nm was read at designated time points.Forty-eight h after transfection, BC cell line MCF-7 suspended in DMEM plus 10% FBS was added into 96-well plates (2  × 102 cells/well) and incubated in DMEM with 10% FBS for 2 weeks. Colonies were subjected to fixation with 95% methanol, 10 min of staining with 0.1% crystal violet, and rinsing with PBS, followed by cell colony counting.Cells in varying transfection groups were treated with 0.25% trypsin, inoculated into 6-well plates (4  × 105 BC cells (MCF-7) were placed into 6-well plates, and a pipette tip (200 µL) was employed to make a scratch through center of each well when cells grew to 80% confluence. Dispersed cells were discarded. Fresh mediums were employed to continuously culture the remaining cells. The migrated cells were observed and pictured at 0 and 24 h. Image J software was used for analysis. Migration width = wound healing width at 24 h –wound healing width at 0 h.After transfection, 1  × 105 cells/mL) and were filled into the upper chamber, while medium with 15% FBS was filled into lower chamber. After being cultured for 48 h at 37 °C, cells that did not pass membranes were removed using a cotton swab, while cells in lower chamber were subjected to 0.1% crystal violet for staining. A microscope was implicated to random 4 fields, and invaded cells were counted and photographed.First, 9.6 mg/ml Matrigel matrix  diluted with serum-free medium at 1:8. Fifty µL of dilution was added to the Transwell upper chambers for 30 min. After transfected for 48 h, BC cells were suspended in 200 µl serum-free DMEM , and protein concentration was assessed by Pierce BCA  protein assay kit. Total proteins were isolated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich). After blocking, membranes were cultivated overnight with primary antibodies, including rabbit anti-CDC25A and rabbit anti-GAPDH. Subsequently, PBS + 0.1% Tween-20 (PBST) was taken to rinse membranes 3 ×10 min. Afterward, secondary antibody goat anti-rabbit IgG H&L (HRP) (ab205718) was incubated with membranes at room temperature for 1 h, and then membranes were rinsed with PBST 3 ×10 min. All protein bands were visualized by chemiluminescence reaction , followed by analysis by Image Lab (Bio-Rad). All antibodies were from Abcam (China).Vectors psiCHECK  fused with mutant-type (MUT) and wild-type (WT) CDC25A 3′ UTR were generated. MCF-7 cells were planted onto 48-well plates for 24 h of culture at 37 °C. miR-100-5p mimic/mimic NC and CDC25A-psiCHECK WT/MUT were co-transfected into cells. Forty-eight h later, the luciferase activity was assayed by Dual-Luciferase Reporter Assay System .BC cells in logarithmic growth phase were transfected. Forty-eight h later, cells were collected. Cell suspension (100 µL) was incubated 15 min in dark with Annexin-V-FITC , and then propidium iodide  was utilized for cell staining. FACS Calibur  was adopted to measure cell apoptosis. Flowjo software  was recommended for data analysis.t-test was used for significance test between the two groups. P < 0.05 was accepted as significant.All data were processed by GraphPad Prism 6.0 . Each experiment was repeated in triplicate, including 3 technical replicates and 3 biological replicates. The results were presented by mean ± standard deviation (SD). To compare differences among three or more groups, one-way analysis of variance, followed by Bonferroni test was used. Student’s P < 0.05)  . Thus, wP < 0.05) (P < 0.05)  (P < 0.05) (P < 0.05)  . CCK-8 a < 0.05) . As unve < 0.05) . Results < 0.05)  and 2E.  < 0.05) . It was P < 0.05) (P < 0.05) but had no influence on that of CDC25A-MUT (P < 0.05) (P < 0.05)  . In BC c < 0.05) . We also < 0.05) . Dual-luC25A-MUT . qRT-PCR < 0.05) . Western < 0.05) . It was P < 0.05), and notably downregulated when miR-100-5p was overexpressed (P < 0.05). In comparison to miR-mimic+oe-NC group, CDC25A was upregulated in miR-100-5p mimic+oe-CDC25A group (P < 0.05) (P < 0.05), while upregulating miR-100-5p could notably suppress this ability(P < 0.05). In comparison to miR-mimic+oe-NC group, cell proliferative ability increased when CDC25A and miR-100-5p were simultaneously overexpressed (P < 0.05) (P < 0.05), while upregulating miR-100-5p could remarkably repress these abilities (P < 0.05). In comparison to miR-mimic+oe-NC group, these abilities were clearly enhanced after CDC25A and miR-100-5p were simultaneously overexpressed (P < 0.05) (P < 0.05), while miR-100-5p upregulation could dramatically facilitate cell apoptosis (P < 0.05). In comparison to miR-mimic+oe-NC, cancer cell apoptotic rate was restrained after CDC25A and miR-100-5p were simultaneously overexpressed (P < 0.05) , miR-NC+oe-CDC25A (CDC25A overexpression group), miR-mimic+oe-NC (miR-100-5p overexpression group), and miR-mimic+oe-CDC25A (simultaneous enforced expression of miR-100-5p and CDC25A group). qRT-PCR and western blot result found that CDC25A mRNA and protein levels were markedly up-regulated in oe-CDC25A group ( < 0.05)  and 4B,  < 0.05)  and 4D.  < 0.05)  and 4F.  < 0.05) . In concWith 1.3 million cases increasing annually, BC is a symbolic cancer in female’s daily life . A recenvia EMT suppression (Present studies disclosed that miR-100-5p is aberrantly expressed in several cancers, modulating cancer cell malignant behaviors. For instance, miR-100-5p is lowly expressed in prostate cancer cells, and it down-regulates mTOR to hamper progression of prostate cancer cells . In chorpression . Wang, Tpression  elucidatin vitro and in vivo by targeting CDC25A (To further elucidate modulatory mechanism of miR-100-5p in progression of BC, CDC25A was predicted to be a target of miR-100-5p, which was unveiled to be highly expressed in BC through bioinformatics analysis. CDC25A is a key regulatory factor in progression of cell cycle and checkpoint response . miR-365g CDC25A . miR-122g CDC25A . miR-449g CDC25A . Here, wIn conclusion, we validated the inhibitory effect of miR-100-5p on malignant behaviors of BC cells. Besides, miR-100-5p suppresses development of BC through reducing CDC25A expression. Our study helps us to gain more awareness of role of miR-100-5p in BC, and brings additional insight into the exploration of novel targeted therapy for BC. Nevertheless, there are several limitations. For instance, it is necessary to further investigate the involvement of signaling pathways downstream of miR-100-5p/CDC25A. Besides, direct relationship of miR-100-5p and CDC25A, as well as their molecular mechanism in regulating biological behaviors are still elusive. These shortcomings need to be resolved in further studies.10.7717/peerj.12263/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12263/supp-2Supplemental Information 2Click here for additional data file.


The electronic spectra indicated that all the isolated complexes have an octahedral structure. The thermal gravimetric analyses confirmed the suggested formula and the presence of coordinated water molecules. The XRD pattern of the metal complexes showed that both Co(II) and Ni(II) have amorphous nature, while Zn(II) complex has monoclinic crystallinity with an average size of 9.10 nm. DFT modeling of the ligand and complexes supported the proposed structures. The calculated HOMO-LUMO energy gap, ΔEH-L, of the ligand complexes was 1.96–2.49 eV range where HAAT < Zn(II) < Ni(II) < Co(II). The antioxidant activity investigation showed that the ligand and Zn(II) complex have high activity than other complexes, 88.5 and 88.6%, respectively. Accordingly, the antitumor activity of isolated compounds was examined against the hepatocellular carcinoma cell line (HepG2), where both HAAT and Zn(II) complex exhibited very strong activity, IC50 6.45 ± 0.25 and 6.39 ± 0.18 μM, respectively.The newly synthesized Schiff's base derivative, N-allyl-2-(2-oxoacenaphthylen-1(2H)-ylidene)hydrazine-1-carbothioamide, has been characterized by different spectral techniques. Its reaction with Co(II), Ni(II), and Zn(II) acetate led to the formation of 1 : 1 (M:L) complexes. The IR and NMR spectral data revealed keto-thione form for the free ligand. On chelation with Co(II) and Ni(II), it behaved as mononegative and neutral tridentate via O, N Schiff bases are a class of compounds that contain azomethine bond, -C=N-, which results from the condensation of active carbonyl compounds with a primary amine. The Schiff bases are very versatile where the most common Schiff bases have NO donor atoms, but in many cases, the oxygen atom may be replaced by sulfur atoms or added to the donor atoms, NS or NSO, respectively . They arThiosemicarbazones, as a subclass of the Schiff bases, are formed by the reaction of ketone or aldehyde and thiosemicarbazide. Thiosemicarbazones and their complexes have noteworthy significance in biological and chemical studies –13. Thei2H groups in addition to both oxygen and sulfur atoms. The APTH was employed as a chelating agent in cloud point extraction procedure of trace amounts of Hg(II) and Cd(II) ions from aqueous medium . Dark brown, yield 70%, m.p 210 °C, Anal. Calc. % for C20H21N3O4SCo (458.40): C 52.40; H 4.62; N 9.17; M 12.86. Found%: C 52.12; H 4.38; N 8.97; M 12.73. Λm (DMSO Ω−1·cm2·mol−1) 9.8. IR : 3314, 1725, 1703, 1664, 1586, 890. UV-Vis : 34970, 32680, 27780, 19160, 17010, 15085.[Ni(HAAT) (OAc)2(H2O)]. Brown, yield 73%, m.p 220°C, Anal. Calc. % for C20H21N3O6SNi (490.16): C 49.01; H 4.32; N 8.57; M 11.97. Found%: C 48.74; H 4.11; N 8.33; M 12.01. Λm (DMSO Ω−1·cm2·mol−1) 10.2. IR : 3420, 3336, 3279, 1702, 1652, 1619, 930. UV-Vis : 34965, 32260, 27935, 20160, 19050, 14750.[Zn(HAAT) (OAc)2]. Brownish yellow, yield 86%, m.p 230°C, Anal. Calc. % for C20H19N3O5SZn (478.38): C 50.17; H 4.00; N 8.78; M 13.65. Found%: C 49.93; H 3.89; N 8.45; M 13.39. Λm (DMSO Ω−1·cm2·mol−1) 10.3. IR : 3323, 3264, 1722, 1677, 1656, 1568, 935. UV-Vis : 35210, 32465, 27930, 24390, 21740, 20750.The ligand and complexes' geometry optimization was performed via the Gaussian 09W suite program  at DFT/BThe antioxidant activity of the ligand and its complexes was carried out using the ABTS assay procedure , 38 in wThe hepatocellular carcinoma cell line (HepG2) was used to investigate the anticancer activity by the well-known MTT assay established on the change in color from yellow to purple due to the conversion of tetrazolium bromide (MTT) to formazan derivative in viable cells, and the relative cell viability % was calculated , 40. The−1·cm2·mol−1  spectral data revealed that HAAT chelated with the metal ion in a mononegative tridentate fashion via the carbonyl oxygen, azomethine nitrogen, and sulfur atom of deprotonated thiol (SH) (−1 due to ν(N4H) vibrations  complex displayed two bands at 3336 and 3279 cm−1, assigned to the ν(N4H) and ν(N2H) vibrations  complex presented ν(N4H), ν(N2H), and ν(C=S) vibrational bands at 3323, 3264, and 935 cm−1  (M. Wt. = 458.40). The observed peak at m/z = 412.02 (1.92%) was attributed to the fact that the complex lost ethanol molecule leading to [Co(AAT)(OAc)]•+ formula, C18H15CoN3O3S•+ (F. Wt. = 412.33). The spectrum displayed another peak at m/z = 368.43 (2.18%) assigned to the C15H7CoN3O3S•+ formula (F. Wt. = 368.23) that resulted from the degradation of the ligand and acetate ion by losing the C3H8 fragment.On the other hand, the Co(II), Ni(II), and Zn(II) complexes' mass spectra showed quite a lot of peaks where the most important one was the molecular ion peak that was observed at 458.37 (2.08%), 490.04 (3.54%), and 478.17 (0.75%), respectively, coincided with the proposed formulae of the complexes Figures –S6. For −1 with a shoulder at 32890 cm−1 attributable to the π ⟶ π∗ transition of aromatic acenaphthaquinone moiety, carbonyl group, and both of azomethine and thione groups [−1 with two shoulders at 22830 and 25000 cm−1 assigned to the n ⟶ π∗ transitions of thione, azomethine, and carbonyl groups [The electronic spectrum of the ligand, in DMF, showed two bands at 35460 and 28570 cme groups , respectl groups , 48, res−1 with a shoulder at 15085 cm−1 designated to 4T1g(F) ⟶ 4T1g(P) (υ3) and 4T1g(F) ⟶ 4A2g(P) (υ2) transitions [7-system parameters, υ1, B, and 10Dq, were calculated via the spin allowed transitions, υ3 and υ2, and were 7049, 729, and 8020 cm−1, respectively, confirming the octahedral arrangement [−1 attributed to the charge transfer from ligand to metal (LMCT) and n ⟶ π∗ transition of the newly formed azomethine group (C=N2) confirming the ligand existence in thiol form  complex spectrum, in DMF, exhibited a band at 17010 cmangement , 50. Furiol form . The shietal ion , 51. Finetal ion , 50, 51.−1 with a shoulder at 32260 cm−1 attributed to the intraligand transitions, (π⟶π∗)Acen, (π⟶π∗)CN&CS, and (π⟶π∗)CO [−1 were assigned to n⟶π∗ transitions of the carbonyl, azomethine, and thione groups [−1 with two shoulders at 19050 and 14750 cm−1 ascribed to the LMCT, 3A2g⟶3T1g(F) (ν2) and 3A2g(F)⟶3T2g(F) (ν1) transitions, respectively, suggesting that the Ni(II) complex has an octahedral geometry [8-system were employed to determine ν3, B, and Dq parameters and were 31195, 399, and 1476 cm−1, respectively. The magnetic moment value was 3.09 B.M. supporting the suggestion of an octahedral structure with 3A2g ground term [The Ni(II) complex displayed two bands at 34965 and 27935 cm(π⟶π∗)CO , 48, rese groups , 48, rese groups . Further−1 with a shoulder at 32265 cm−1 attributed to the intraligand transitions, CN&CS, and (π⟶π∗)CO [−1 with shoulders at 24390, 22420, and 21740 cm−1 was assigned LMCT, (n⟶π∗)CO, (n⟶π∗)CN, and (n⟶π∗)CS transitions [n⟶π∗ transition bands were shifted to higher or lower wavenumbers supporting their involvement in the metal ion chelation [Finally, the Zn(II) complex displayed two bands at 35210 and 27930 cm(π⟶π∗)CO , 48, resnsitions , 48, reshelation .2O and/or EtOH molecules whichever outside or inside the chelation sphere.The thermogravimetric analyses of the ligand and isolated solid complexes confirmed the existence of H4H6NS , while the second step corresponded to the complete decomposition of the ligand  leading to a carbon ash residue   .The Co(II) and Zn(II) complexes' TG curves displayed only two successive decomposition stages over 125–445 and 445–610°C ranges. The first stage of the Co(II) complex was due to the loss of coordinated ethanol molecule and acetate anion  while that of Zn(II) was due to the loss of the acetate anions . The second stage in both complexes was attributed to complete decomposition of the ligand leading to a residue of metallic residue  and Zn(II) complexes, respectively) .3H6N moiety . The third degradation step was extended from 455°C to 640°C and attributed to complete decomposition of the ligand  leaving a metallic residue of nickel metal   complex showed that it has thermal stability up to 155°C at which the first decomposition step was observed and attributed to the loss of the coordinated water molecule and acetate anions . The second step was observed over the 390–455°C range and corresponds to the loss of C 11.97%) .θ) at wavelength of 1.54 Å to investigate their crystallinity degree. The diffraction patterns of both Co(II) and Ni(II) complexes showed no significant peaks, and the trend of the curves decreases from maximum to minimum intensity indicating the amorphous nature of the complexes [Unfortunately, several trials to get crystals of the newly synthesized metal complexes suitable for single-crystal studies did not succeed which may originate from the fact that they were soluble only in DMSO and DMF. So, the powder X-ray diffraction patterns of Co(II), Ni(II), and Zn(II) complexes were scanned in the range of 5–80  and miller indices (hkl), the lattice parameters of monoclinic (2) powder were evaluated from the peak position using the following relation [On the other hand, the powder X-ray diffraction pattern of [Zn(HAAT) (OAc)d 2.16 Å . Powder  γ = 90° . The calrelation , 55:(1)1D, was evaluated using Debye-Scherrer's equation [D = 0.9 λ/βcosθ, where λ is the X-ray wavelength and β is the full width at half maximum intensity (FWHM). The obtained crystalline size was in the range 4.19–16.34 nm exhibiting an average size of 9.10 nm. The microstrain or lattice strain, ε, is a measure of the deviation from reference lattice positions and may attribute to the defects and dislocations at the grain boundaries. The lattice strain was determined from the relation, ε = β/4tanθ [−3, which denotes high lattice strain. Moreover, the dislocation density, δ, which refers to the crystal imperfection, was determined from Williamson and Smallman's relation, δ = 1/D2 [−3 nm with an average value of 24.84 × 10−3 nm which reflects the remarkable change of the grain size with values of 2θ of the complex.The crystalline size of the Zn(II) complex, equation , D = 0.9 β/4tanθ , 58, andδ = 1/D2 , and wasEg) of the ligand and complexes from their absorption spectra, αhν = A (hν -Eg)r, where A and r are independent constants. The r value for the indirect transition was 1/2 while, for direct transition, it equals 2 [hν, in the plot of (αhν)r against (hν) at different r values, established by linear portion extrapolating, represents the optical band gap (Eg).Tauc's equation was applied to estimate the optical band gap  complex has the lowest one, 2.18 eV  atom slightly deviated from the acenaphthaquinone plane where Cnph-n-Cnph-o-O-Co and Cnph-o-Cnph-n-N1-Co angles were 19.27 and -8.08°, respectively. The bond angle data cleared that the complex has a small degree of distortion as, for example, the O-Co-N1, S-Co-N1, and O-M-S were 92.30, 86.94, and 97.02°, respectively, which are higher or lower than the ideal value (90°). Furthermore, another type of distortion was observed from the bond length data as the Co-S, 2.24 Å, was longer than M-N1 and M-O, 1.86 and 1.92 Å, respectively, but compatible with the corresponding X-ray values [On the other hand, the DFT optimized structure Co(II) complex revealed octahedral geometry around the metal atom in which the ligand has an angular configuration . The dihy values , 67  complex, where the angle between the carbonyl oxygen and azomethine nitrogen, O-Cnph-o-Cnph-n-N1, was -5.80° while the thiocarbonyl carbon and N2H nitrogen atoms were tilted on the acenaphthaquinone plane as CS-N2-N1-Cnph-n and Cnph-o-Cnph-n-N1-N2 were -158.32 and 127.30°, respectively. The Ni(II) atom deviated from the acenaphthaquinone plane where the Cnph-n-Cnph-o-O-Ni and Cnph-o-Cnph-n-N1-Ni angles were 14.34 and -6.51°, respectively. Likewise, the bond lengths exhibited another type of distortion as the Ni-S bond was longer than M-N1 and M-O bonds by ∼ 0.5 Å but all were in accordance with the similar complexes reported previously [As Co(II) complex, the Ni(II) complex exhibited less bond angle distortion from ideal values of the octahedral structure ; e.g., Oeviously , 67  atom slightly deviated from the acenaphthaquinone plane where the Cnph-n-Cnph-o-O-Zn and Cnph-o-Cnph-n-N1-Zn angles were 0.15 and 0.82°, respectively. Likewise, the Zn(II) complex exhibited small bond length distortion as Zn-N1 was longer than Zn-O bonds by 0.04–0.05 Å and was in agreement with the X-ray reported values [Moreover, in Zn(II) complex, the dihedral angles indicated that the carbonyl oxygen, azomethine nitrogen, and Ne moiety , where Od values , 67 (TabH and EL) while the Zn(II) complex exhibited the highest energies. The chemical stability and intramolecular charge transfer may be correlated with the HOMO-LUMO energy gap, ΔEH-L, where the ΔEH-L decrease results in more feasible charge transfer which is one of the significant factors affecting the molecule bioactivity [H-L gap ranging from 1.96 to 2.49 eV and may be ordered as HAAT > Zn(II) > Ni(II) > Co(II)  by only 0.05–0.60 eV.On the other hand, the energies of the frontier molecular orbitals, HOMO and LUMO, were determined where they act as electron donor and accepter, respectively. activity –70. The > Co(II) . MoreoveEH and EL values were employed to determine some chemical reactivity descriptors like electronegativity (χ), global hardness (η), softness (δ), and electrophilicity (ω) using the following equations [χ, and η, respectively. The ligand has the highest electronic acceptability, softest, and electrophilicity that measure the energy reduction that originated from HOMO-LUMO electron flow, high δ, and ω, respectively.Finally, the obtained quations  (Table 6Antioxidants' scavenge reactive oxygen species (ROS), such as superoxide, hydroxyl, and hydrogen peroxide radicals, cause many life-threatening diseases. Human diseases, like cancer, diabetic mellitus, hypertension, and aging, may be initiated by the ROS capability to destruct DNA, proteins, and membrane functions. The ROS are a by-product of normal metabolism in different subcellular compartments, even under optimal circumstances –73.The ligand and complexes were examined as an antioxidant by the ABTS method in which L-ascorbic acid was used as a standard material. The data showed that the ligand and Zn(II) complex have high activity than other complexes and close to the ascorbic acid, 88.5 and 88.6%, respectively . The Ni. CompariFinally, the L-ascorbic acid antioxidant activity may originate from its action as a reducing substance; i.e., it donates high-energy electrons to neutralize free radicals . Thus, t50 6.45 ± 0.25 and 6.39 ± 0.18 μM, respectively, comparable to the doxorubicin standard, 4.50 ± 0.2 μM. The Ni(II) complex has moderate activity, IC50 21.46 ± 0.72, while the Co(II) complex has weak cytotoxic activity, weak activity 67.31 ± 1.35 μM -ylidene)hydrazine-1-carbothioamide, revealed that it is in thione form. The HAAT formed 1 : 1 (M:L) octahedral complexes with Co(II), Ni(II), and Zn(II) acetates. The ligand chelated with Co(II), Ni(II), and Zn(II) ions as ONS donor in mononegative and neutral tridentate in Co(II) and Ni(II) complexes, respectively, while in Zn(II) complex, it acts as neutral bidentate, via ON atoms. The DFT calculations showed that the ligand has a planar structure while it has bent conformation reflecting its flexibility. The antioxidant activity investigation revealed that the ligand and Zn(II) complex have high activity almost equal to the standard material, ascorbic acid. Similarly, the MTT assay was utilized for the examination of the antitumor activity using the hepatocellular carcinoma cell line (HepG2). The data revealed that the ligand and Zn(II) complex presented very strong activity.


The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some β-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.Four NDM-1 mutants  were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some β-lactams. Significant reduction of k Each kinetic value represents the mean of the results of three different measurements; the error rate was below 10%. The molar extinction coefficients and wavelengths used in the assay were imipenem ΔεM300, −9000 M−1 cm−1; meropenem ΔεM297, −6500 M−1 cm−1; cefepime ΔεM260, −10,000 M−1 cm−1; cefazolin ΔεM260, −7400 M−1 cm−1; cefoxitin ΔεM260, −7775 M−1 cm−1; cefotaxime ΔεM260, −7500 M−1 cm−1; ceftazidime ΔεM260, −7500 M−1 cm−1; carbenicillin ΔεM235, −780 M−1 cm−1; and benzylpenicillin ΔεM235, −775 M−1 cm−1; nitrocefin ΔεM482, −15,000 M−1 cm−1.Steady-state kinetic parameters, Kequation . Reactiois known . The eff2, pH 7.0. The excitation wavelength was 280 nm and the emission spectra, in the range of 300–500 nm, were recorded at 25 °C.Fluorescence studies were carried out on a Perkin-Elmer LS-50B spectrofluorometer. The protein concentrations were 8 µg/mL. The buffer used was 20 mM Hepes buffer, 20 µM ZnCl2. The plate was heated from 25 °C (2 min) to 99 °C (2 min) with heating rate of 1 °C/min. The fluorescence intensity was measured with Ex/Em:490/530 nm. Analysis of Boltzmann Tm (TmB) and derivative Tm (TmD) was carried out by Protein Thermal Shift Software Version 1.4. The melting temperature TmB was calculated by fitting data in the region of analysis to the Boltzmann equation  whereas TmD was calculated for the region of analysis using the derivative of the melt curve .The thermal stability of NDM-1, L218T, L221T, L269H, Y229W and L218T/Y229W was determined using a fluorescence-based thermal stability assay  in a 7500 Fast Real-Time PCR System . The protein melt reaction mix  was added to the wells of the 96-well PCR plate. The melt reaction mix included 1 μg of each enzyme, protein shift dye (8×) and 20 mM Hepes pH 7 + 20 μM ZnClcal/mol/Å2 gradient with AMBER10:EHT force field was minimised. A time step of 1.0 fs was employed after an initial energy minimisation of 100 ps; the system was gradually heated from 10 to 330 K in 100 ps. The simulations were performed in the NVT ensemble (T = 300 K) for 100 ps and NPT ensemble (T = 300 K) at a constant pressure of 1 bar for 200 ps. The production run for MD simulations was carried out for 20 ns for both free enzymes and enzyme-ligand complex. The latter was obtained from the selected enzyme and the hydrolysed meropenem (retrieved from 5YPM structure) [Molecular dynamics simulations (MDs) were carried out using MOE software 2020.09  . The cryructure) . In this


Observed variations in coordination number are ascribed as being related to chloride solvophobicity. Optical spectra are presented, while magnetism measurements indicate governance of the magnetism by zero-field splitting of the cobalt ion.With cobalt(II) chloride, some piperazine- and homo trans-2,5-dimethyl-1,4-bis[2-(pyridin-2-yl)ethyl]piperazine (Pdmpz) and tridentate 4-methyl-1-[2-(pyridin-2-yl)ethyl]homopiperazine (Pmhpz). The CoCl2 complexes with Ppz, namely, {μ-1,4-bis[2-(pyridin-2-yl)ethyl]piperazine}bis[dichloridocobalt(II)], [Co2Cl4(C18H24N4)] or Co2(Ppz)Cl4, and Pdmpz , are shown to be dinuclear, with the ligands bridging the two tetrahedrally coordinated CoCl2 units. Co2(Ppz)Cl4 and {dichlorido{4-methyl-1-[2-(pyridin-2-yl)ethyl]-1,4-diazacycloheptane}cobalt(II) [CoCl2(C13H21N3)] or Co(Pmhpz)Cl2, crystallize in the monoclinic space group P21/n, while crystals of the pentacoordinate monochloro chelate 1,4-bis[2-(pyridin-2-yl)ethyl]piperazine}chloridocobalt(II) perchlorate, [CoCl(C18H24N4)]ClO4 or [Co(Ppz)Cl]ClO4, are also monoclinic (P21). The complex {1,4-bis[2-(pyridin-2-yl)ethyl]-1,4-diazacycloheptane}dichloridocobalt(II) [CoCl2(C19H26N4)] or Co(Phpz)Cl2 Cl2, [Co(Ppz)Cl]+ and Co(Pmhpz)Cl2 have substantial trigonal–bipyramidal character in their stereochemistry. Visible- and near-infrared-region electronic spectra are used to differentiate the two types of coordination spheres. TDDFT calculations suggest that the visible/NIR region transitions contain contributions from MLCT and LMCT character, as well as their expected d–d nature. For Co(Pmhpz)Cl2 and Co(Phpz)Cl2, variable-temperature magnetic susceptibility data were obtained, and the observed decreases in moment with decreasing temperature were modelled with a zero-field-splitting approach, the D values being +28 and +39 cm−1, respectively, with the S = 1/2 state at lower energy.Syntheses are described for the blue/purple complexes of cobalt(II) chloride with the tetradentate ligands 1,4-bis[2-(pyridin-2-yl)ethyl]piperazine (Ppz), 1,4-bis[2-(pyridin-2-yl)ethyl]homopiperazine (Phpz),  The distances are unexceptional for CoII to tertiary amine linkages  bond lengths Tables 1 ▸ ▸ ra2-Ppz combination, the dinuclear compound Co2(Ppz)Cl4 was obtained Clet al., 2012et al., 2017abvs the larger ionic radius of Co2+vs Cu2+/Ni2+. This proposal was approached by synthesising the homopiperazine analogue, Phpz, whose ligand has a larger (N2—N2A) bite. The compound Co(Phpz)Cl2 was indeed obtained as a mononuclear product . However, anticipatedly quadridentate Phpz is now seen to act as a tridentate ligand, with the cobalt(II) ion being pentacoordinate.As the same ligand behaves as a straightforward mononucleating quadridentate in the copper and nickel complexes  Cl](BPh4) . We hence prepared the compound of composition [Co(Ppz)Cl]ClO4, thus removing a chloride from the binding competition. The resulting structure bears out this hypothesis Cl)]+ cation. This structure has τ = 0.65, so is substantially trigonal–bipyramidal in its coordination geometry; the reference axis is Co1A–Cl1A, and the (pseudo)trigonal axis is N2A–Co1A–N4A. The cation is asymmetric, with non-matching Co—Npyridine bonds of 2.057 (5) and 2.109 (5) Å, while the Co—Namine distances are notably inequivalent, at 2.098 (5) for Co1A—N3A, but 2.238 (5) Å for Co1A—N2A – the longest Co—N bond in this set of four compounds. One might note that N3A is ‘trigonal–equatorial’, vs N2A being ‘trigonal–axial’, and suspect that this longer bond betokens an instability that leads to Co2(Ppz)Cl4. The perchlorate may be involved with quite weak C—H⋯O hydrogen-bonding interactions: e.g., C11A⋯O3B, C13A⋯O4B, and C11A⋯O4C are 3.28, 3.46 and 3.60 Å, respectively.[Co(Ppz)Cl)]ClO2 Cl2 Fig. 5 crystallτ = 0.57 and the reference axis being Co1–Cl1. The sole pyridine nitrogen N3 and the methylated piperazine nitrogen N1 form the pseudo-trigonal axis. Analogously to the [Co(Ppz)Cl)]+ situation, the pseudo-equatorial Co—N2amine bond, at 2.097 (4) Å, is shorter that the Co—N1amine [2.232 (5) Å] and Co—N3pyridine [2.146 (4) Å] bonds in the trigonal directions. One may note that the same axial vs equatorial Co–N bond-length relationship also holds for Co(Phpz)Cl2, above.The coordination core is somewhat trigonal–bipyramidal, with Electronic spectra: Pseudotetrahedral species: The essentially identical UV–Vis–NIR spectra for [Co2(Ppz)Cl4] and Co(Pdmpz)Cl2 Cl4] is ultimately confirmed by the elemental analyses (vide infra).l2 Fig. 6 strongly4en)]Cl2, which has maxima at ca 1670, 1380, 1000, 650 and 580 nm, attributed in a crystal-field model to 4A2 → 4T1 (F) transitions (the first three) . Though shifted slightly, these maxima are quite similar to the bands for [Co2(Ppz)Cl4] and [Co2(Pdmpz)Cl4]. The DFT results for a CoN2Cl2 chromophore of Co2(Ppz)Cl4 suggest that even the low-energy transitions involve CT contributions from the CoCl2 moiety to the pyridine ring Cl4] and other CoN2Cl2 chromophores, the roughly trigonal–bipyramidal archetypal CoN3Cl2 systems Co(Me5dien)Cl2 and [Co(Et4dien)Cl2] also have strong ligand-field absorptions in the visible region near 500 and 650 nm, as well as NIR bands at ca 2500, 1140, and 950 nm  and 4E(P) Cl]ClO4, respectively. In comparison with the CoN2Cl2 cores, one should note the rather different pattern of absorption bands in the NIR. Firstly, the band near 1000 nm appears to be supplanted by two bands, one being near 750 nm, the other around 950 nm. More tellingly, the 1100–1500 nm region, which has clear CoN2Cl2 maxima near 1300 and 1700 nm, becomes hollowed out, and broader features appear at 1600–1900 nm. The Vis–NIR spectrum  of Co(Pmhpz)Cl2 is, as expected, similar to that of Co(Phpz)Cl2. We do note that the utility of NIR spectroscopy for tetra- and pentacoordinate cobalt(II) complexes, pioneered by Goodgame & Goodgame (1965Table S1).Figs. 8game 1965 has hardMagnetism analysiset al., 2016et al., 20182 and Co(Phpz)Cl2  complexes that have recently been discovered to act as single-ion/single-molecule magnets (SIM/SMM) at reduced temperature . We were able to fit the data through most of the temperature regime and the extracted D, gave, Δ, a and b which are listed in Table 6via:x and χz are the longitudinal and transverse modes of the anisotropic responses (Δ = xS/zS), a is the TIP and b the total diamagnetic correction.The magnetism as a function of temperature and applied field showed no evidence for SMM behaviour. In situations like this, the temperature dependence of the moments has been recognized as being due to zero-field splitting . The D and gave values appear to be in the normal ranges; D values for CoII do cover a wide range, from ca −50 to +100 cm−1  term, and the anisotropy values also confirm the findings as self-consistent , likely of little energetic consequence.There are no true supramolecular structures formed by the compounds, whose crystal lattices containing individual molecules are defined mainly by weak, non-bonding interactions. Along with the absence of any solvation of these crystals, the only hydrogen-bonding interactions observed are in [Co(Ppz)Cl]ClOsupporting information .Some lattice views of the compounds are displayed in the M(pyridylethylpiperazine)X2, M(pyridylethylpiperazine)X+, M(pyridylethylhomopiperazine)X2 or M(pyridylethylhomopiperazine)X+ structures include [Co(Phzp)Cl]BPh4 , while structural diagrams were generated using the CrystalMaker-10 software and Preview-10. Reagents were used as received from TCI America, Sigma–Aldrich, MCB and Fisher Scientific. Elemental microanalyses were by Robertson Microlit Laboratories .Chemical ionization mass spectra were obtained on a Thermo-Electron LTQ–FT 7T FT–ICR instrument. UV–visible–near infrared spectra were obtained using PerkinElmer Lambda-35 or Shimadzu UV3600Plus spectrophotometers equipped with integrating spheres for solid-state spectroscopy. Magnetic susceptibility data between 1.8 and 310 K in an applied field of 1 kOe were collected using a Quantum Design MPMS-XL SQUID magnetometer. Crystals were powdered and packed into #3 gel capsules that were placed inside drinking straws attached to the sample rod. The magnetization was measured at 1.8 K as a function of increasing field from zero to five tesla and at selected fields returning to zero. The data were corrected for the contributions from the sample holders (measured independently) and the diamagnetism of the constituent atoms, as estimated using Pascal’s constants ethyl]piperazine (Ppz): A mixture of piperazine , 2-vinylpyridine , and 2 drops of glacial acetic acid was set to react at ca 368 K for 14 to 50 h in a capped tube. The reaction mixture was allowed to cool to room temperature, resulting in the formation of a brown solid mass. The mass spectrum indicated Ppz as the dominant component of the solid: m/z = 297.207, calculated for (C18H24N4+H)+, 297.208. The crude ligand was used without purification in the synthesis of the cobalt complexes.1,4-Bis[2-(pyridin-2-yl)ethyl]homopiperazine (Phpz): From 2-vinylpyridine  and homopiperazine ; crude ligand as a brown mass; m/z = 311.223, calculated for (C19H26N4+H)+, 311.224.trans-2,5-Dimethyl-1,4-bis[2-(pyridin-2-yl)ethyl]piperazine (Pdmpz): From trans-2,5-dimethylpiperazine  and 2-vinylpyridine  as a brown solid mass mingled with white crystals. m/z = 325.239, calculated for (C20H28N4+H)+, 325.239.4-Methyl-1-[2-(pyridin-2-yl)ethyl]homopiperazine (Pmhpz):N-methylhomopiperazine  and 2-vinylpyridine : heated at the boiling point (ca 433 K) for 3 min.; as a viscous brown oil; m/z = 220.181, calculated for (C13H21N3+H)+, 220.181Synthesis of cobalt complexes: The cobalt(II) compounds were mainly prepared by the general method exemplified for [Co2(Ppz)Cl4] below, using amounts of crude ligands equivalent to the molecular content of the diazacycloalkane used for the ligand synthesis.2(Ppz)Cl4]:::ClO The blue crystals produced were filtered off and recrystallized from acetonitrile. MS m/z = 390.102 (M − ClO4)+ = C18H24N4CoCl+, calculated 390.102. Analysis C,H,N: found %, C 44.3, H 4.78, N 11.4; calculated for C18H24N4CoCl2O4, C 44.1, H 4.93, N 11.4.2(Pdmpz)Cl4]:[Co The blue crystals produced were filtered off and recrystallized from nitromethane. MS m/z = 454.111, (M + H)+: calculated for C20H29Cl2Co2N4+, 454.110; m/z = 418.133, (M − Cl)+, calculated for C20H28ClCo2N4+, 418.133. Analysis C,H,N: found %, C 41.6, H 4.80, N 9.44; calculated for C20H28Cl4Co2N4: C 41.1, H 4.83, N 9.59.via ω-scans using an Atlas CCD detector using Cu Kα radiation or a Bruker AXS D8 Quest diffractometer with a PhotonII charge-integrating pixel array detector (CPAD). Data for those structures were collected, scaled and corrected for absorption using the CrysAlis PRO 2015 software suite program package . The SQUEEZE procedure corrected for 69 electrons within the solvent-accessible voids, or around two nitromethane molecules. The central part of the metal complex (two of the Co-coordinated nitrogen atoms and the C atoms bridging between them) are disordered by a pseudo-mirror operation. Additional disorder that is vaguely recognizable (largest difference peak 0.78 electrons) was ignored. The two disordered moieties were restrained to have similar geometries. Uij components of ADPs for disordered atoms closer to each other than 2.0 Å were restrained to be similar. Subject to these conditions, the occupancy ratio refined to 0.914 (3):0.086 (3).The structure of Co(Phpz)ClUiso(H) = 1.2Ueq(C).For all compounds, H atoms were placed in calculated positions (C—H = 0.95–0.99 Å) and refined as riding with 10.1107/S2056989022001220/yy2006sup1.cifCrystal structure: contains datablock(s) global, CoPhpzCl2_sq, ta-eab1701-c, ta-eab1607, ta-sa15-05. DOI: 10.1107/S2056989022001220/yy2006ta-sa15-05sup2.hklStructure factors: contains datablock(s) ta-sa15-05. DOI: 10.1107/S2056989022001220/yy2006CoPhpzCl2_sqsup3.hklStructure factors: contains datablock(s) CoPhpzCl2_sq. DOI: 10.1107/S2056989022001220/yy2006ta-eab1701-csup4.hklStructure factors: contains datablock(s) ta-eab1701-c. DOI: 10.1107/S2056989022001220/yy2006ta-eab1607sup5.hklStructure factors: contains datablock(s) ta-eab1607. DOI: 2111922, 2111921, 2111920, 2111919CCDC references:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


In the crystal, molecules are connected by C—H⋯π and π–π stacking interactions, forming a layer lying parallel to the ° with the dimethylphenyl ring. The molecular conformation is stabilized by an intramolecular N—H⋯O hydrogen bond with an S(6) ring motif. In the crystal, molecules are connected by C—H⋯π and π–π stacking interactions, forming a layer lying parallel to the (11In the title compound, C Of these, the compound most similar to the title compound is 7-methoxy-3-(2-phenylhydrazinylidene)-1-benzofuran-2(3H)-one, I -N′-[1-(pyridin-2-yl)ethylidene]acetohydrazide, II (TODMEH), 2-(2-nitro-1H-imidazol-1-yl)-N′-[1-(pyridin-2-yl)ethylidene]acetohydrazide, III (TODMIL), 2-(4-nitro-1H-imidazol-1-yl)-N′-[phenyl(pyridin-2-yl)methylidene]acetohydrazide, IV (TODMOR) and 2-(4-nitro-1H-imidazol-1-yl)-N′-[phenyl(pyridin-2-yl)methylidene]acetohydrazide, V (TODMUX). Compound I crystallizes in the monoclinic space group C2/c with Z = 8. In the crystal of I, pairs of molecules are linked into dimers by N—H⋯O hydrogen bonds, forming an a-axis direction. These dimers are connected through π–π stacking interactions between the centroids of the benzene and furan rings of their 2,3-dihydro-1-benzofuran ring systems. Furthermore, there exists a C—H⋯π interaction that consolidates the crystal packing. Compounds II and IV crystallize in the monoclinic space group P21/c with Z = 4. Compound III crystallizes in the monoclinic space group I2/a with Z = 8 and V crystallizes in the triclinic space group PZ = 2. Compound VI crystallizes in the monoclinic space group P21/c with Z = 4. The E conformation in II, III and V is stabilized by a strong intermolecular N—H⋯O interaction. These interactions lead to the formation of dimeric structural arrangements. In the crystal of IV, an intermolecular N—H⋯N hydrogen bond results in a helical chain structure along the b-axis direction. Non-classical intermolecular C—H⋯N and C—H⋯O interactions are also observed in the crystals of II, III, IV and V.A search of the Cambridge Crystallographic Database -3-[2-hydrazinylidene]benzofuran-2(3H)-one was synthesized according to the reported method -2-{[2-hydrazinylidene]methyl}phenol , tetramethylethylenediamine (TMEDA) , CuCl  and CCl4 . After 1–3 h , the reaction mixture was poured into a 0.01 M solution of HCl , and extracted with dichloromethane (3 × 20 mL). The combined organic phase was washed with water (3 × 20 mL), brine (30 mL), dried over anhydrous Na2SO4 and concentrated in vacuo using a rotary evaporator. The residue was purified by column chromatography on silica gel using appropriate mixtures of hexane and dichloromethane (3/1–1/1). Then the substance was refluxed in methanol for 30 min, and left for evaporation. After three days, single crystals of the title compound suitable for X-ray analysis were obtained. Colorless solid (65%); m.p. 475 K. Analysis calculated for C16H14N2O2 (M = 266.30): C 72.17, H 5.30, N 10.52; found: C 72.13, H 5.26, N 10.48%. 1H NMR  δ 12.04 , 6.79–7.69 , 2.37 . 13C NMR  δ 160.47, 157.77, 134.91, 125.14, 124.12, 121.77, 121.56, 119.86, 118.21, 114.55, 108.16, 106.59, 16.85 and 16.52. ESI–MS: m/z: 267.23 [M + H]+.(Uiso(H) = 1.2 or 1.5Ueq(C). The methyl group with the C16 atom attached to the atom C13 is disordered over two orientations, with occupancies of 0.67 (4) and 0.33 (4). Owing to poor agreement, nine reflections  I. DOI: 10.1107/S2056989021011749/is5559Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021011749/is5559Isup3.cmlSupporting information file. DOI: 1983650CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Nature 10.1038/s41586-021-03687-wPublished online 11 August 2011Correction to: Qneo for W7-X discharge 20180918.045,” errors appeared in reference to the configurations shown in the curves of Fig. 3a and associated Fig. 3b−e panels. In the sentence originally reading “This comparison is provided in Fig. 3a for the configurations  W7-X standard,  W7-X high-mirror,  LHD R0 = 3.6 m, and  LHD R0 = 3.75 m....,” the references to Fig. 3b−e should instead have referred to the lettered curves “b,c,d,e” shown in the graph of Fig. 3a. The text has now been amended to read: “This comparison is provided in Fig. 3a for the configurations (b) W7-X standard, (c) W7-X high-mirror, (d) LHD R0 = 3.6 m, and (e) LHD R0 = 3.75 m….”In the original version of this Article, in the fifth paragraph of the section titled “Additionally, a member of the W7-X Team was incorrectly listed as “M. Sanchez”; their name has been corrected to “E. Sánchez.”The original Article has been corrected online.


Yersinia pseudotuberculosis is a foodborne pathogen that subverts immune function by translocation of Yersinia outer protein (Yop) effectors into host cells. As adaptive γδ T cells protect the intestinal mucosa from pathogen invasion, we assessed whether Y. pseudotuberculosis subverts these cells in mice and humans. Tracking Yop translocation revealed that the preferential delivery of Yop effectors directly into murine Vγ4 and human Vδ2+ T cells inhibited anti-microbial IFNγ production. Subversion was mediated by the adhesin YadA, injectisome component YopB, and translocated YopJ effector. A broad anti-pathogen gene signature and STAT4 phosphorylation levels were inhibited by translocated YopJ. Thus, Y. pseudotuberculosis attachment and translocation of YopJ directly into adaptive γδ T cells is a major mechanism of immune subversion in mice and humans. This study uncovered a conserved Y. pseudotuberculosis pathway that subverts adaptive γδ T cell function to promote pathogenicity. Y. pseudotuberculosis preferentially targets an adaptive subset of these cells to subvert immune function. We found that direct injection of Yersinia outer proteins (Yop) into adaptive γδ T cells inhibited their anti-pathogen functions. We screened all Yop effectors and identified YopJ as the sole effector to inhibit adaptive γδ T cell production of IFNγ. We determined that adaptive γδ T cell subversion occurred by limiting activation of the transcription factor STAT4. When we infected mice with Y. pseudotuberculosis expressing an inactive YopJ, this enhanced the adaptive γδ T cell response and led to greater cytokine production from this subset of cells to aid mouse recovery. This mechanism of immune evasion appears conserved in humans as direct injection of Y. pseudotuberculosis YopJ into human γδ T cells inhibited cytokine production. This suggested to us that Y. pseudotuberculosis actively inhibits the adaptive γδ T cell response through YopJ as a mechanism to evade immune surveillance at the site of pathogen invasion.Unconventional γδ T cells are a dynamic immune population important for mucosal protection of the intestine against invading pathogens. We determined that the foodborne pathogen  Yersinia include three species  that can cause human disease. Y. pseudotuberculosis and Y. enterocolitica cause enteric infections .–47.Y. ps T cells , similarrculosis . These dY. pseudotuberculosis adheres to host cells with the bacterial adhesins invasin (Inv) and YadA to translocate effectors through the T3SS [Y. enterocolitica, both Inv and YadA bind β1-integrin either directly or indirectly through the extracellular matrix, respectively [1-integrin expressed on host cells is a known adhesion target for Y. pseudotuberculosis [Y. pseudotuberculosis with a deletion of Inv (ΔInv), YadA (ΔYadA), or both (ΔInv ΔYadA)   were uti with WT . In cont with WT . Thus, YY. pseudotuberculosis can translocate Yop effectors into adaptive Vγ4 T cells, a WT strain expressing a YopE-β-lactamase fusion protein (Yptb-βla) in conjunction with a FRET-based β-lactamase reporter assay was used [+) or lack thereof by emission in the green range (Yop-) [Y. pseudotuberculosis reporter (ΔYopB Yptb-βla) was used as a translocation deficient control. Stimulation of MLN cell suspensions with WT or ΔYopB Yptb-βla confirmed reporter activity at various MOI  ,64,65. Aious MOI . Two houlocation . Yop tra targets . Additio T cells . Y. pseulocation . YadA anintegrin –69. Analintegrin . In cont T cells . Thus, YYptb-βla was utilized to determine whether Vγ1.1/2- CD44hi CD27- γδ T cells that contained Yop effectors were functionally impaired. An MOI of 1 was used as it provided similarly sized populations of Vγ1.1/2- CD44hi CD27- γδ T cells that did or did not contain translocated effectors from the same culture conditions  cultures stimulated with the WT Yptb-βla reporter. Approximately 8% of human Vδ2+ T cells were Yop+ and these cells had significantly reduced IFNγ production as compared to the Yop- counterparts  mutant that lacks acetyl transferase activity, but not other mutant Y. pseudotuberculosis, restored IFNγ production in Vγ1.1/2- CD44hi CD27- γδ T cells , Nkg7 , Prf1 (perforin-1), and Il17a (IL-17A) appear to be regulated directly by translocated YopJ in adaptive Vγ4 T cells  and suggest that translocated YopJ may limit the activation of adaptive Vγ4 T cells. There was also a notable impact (48.0%) on genes associated with cell proliferation, metabolism and energy, mitosis and cell cycle, RNA/DNA processing, and ER/Golgi processing -domain family, Krüppel-like factor and specificity protein (KLF/SP) transcription factor gene family, and the interferon regulatory factors (IRF) family of transcription factors   was able to partially overcome YopJ mediated inhibition, lower levels of IL-12p70 addition (2 and 10 ng/ml) were unable to overcome YopJ mediated inhibition  . Flow cyculosis) . These dn levels . Additiolocation . As IL-1cultures . Thus, cIL-12p70 . Finallyhibition . Importain vivo, foodborne infection with WT and YopJC172AY. pseudotuberculosis was performed on naïve Balb/c mice. As YopJC172AY. pseudotuberculosis is attenuated in vivo [8 CFU) infection dose of YopJC172AY. pseudotuberculosis was administered to normalize the internal bacteria burdens in the MLN between infection groups , Vγ1.1/2- CD44hi CD27- γδ T cells from the MLN of YopJC172AY. pseudotuberculosis infected mice also displayed enhanced IFNγ production compared to their WT Y. pseudotuberculosis infected counterparts , and CD4 and CD8 T cells was performed. Consistent with our in vitro observations, Vγ1.1/2- CD44hi CD27- γδ T cells and myeloid cells contained translocated Yop in vivo  expressing a mutation in the internalin A gene (InlAM) was used for foodborne infection to facilitate epithelial cell invasion [ML. monocytogenes was cultured overnight at 37°C and 220 RPM in BHI media. The following morning, L. monocytogenes was sub-cultured 1:10 in BHI at 37°C and 220 RPM for approximately 2 hours. Infection doses were based on the OD600. Mice were food and water deprived for 4 hours. Approximately 0.5 cm3 bread pieces were inoculated with 2x109 CFU L. monocytogenes in 50 μL. Mice were monitored to ensure the inoculated bread was consumed within 1 hour. Mice that did not fully consume bread were removed from the study. Bacterial infection doses were confirmed by plating inoculum on BHI.invasion . InlAMLY. pseudotuberculosis strains  supplemented with 10% fetal bovine serum, 0.01 M HEPES, 100 μM non-essential amino acids (Gibco), 2 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl or 1x Glutamax (Gibco), and 1 mM sodium pyruvate. Cells were counted using a Vi-CELL Viability Analyzer (Beckman Coulter). Cells were stimulated in 96 well round-bottom tissue culture treated plates with various strains of Y. pseudotuberculosis at 1 or 10 MOI  at 37°C/5% CO2. 100 U/mL of penicillin and 100 μg/mL of streptomycin were added to cells 2 hours post-stimulation. Cells were stimulated for a total of 24 hours or as indicated. BD GolgiPlug (BD Biosciences) was added 5 hours prior to the end of stimulation. If translocation was assessed, β-lactamase Loading Solutions kit (Invitrogen) was used to load CCF4-AM by incubating CCF4-AM at RT with cells for 1 hour in the dark. Cells were then processed for surface staining via incubation with live/dead stain, antibody, and Fc block (BioXcell) for 20 min in the dark at 4°C. Antibodies used included antibodies specific to CD45, CD3, TCRδ, CD8, CD4, Vγ1.1, Vγ2, CD44, CD27, F4/80, CD11b, MHCII, CD11c, and CD29 (BioLegend). Cells were fixed, permeabilized, and stained with anti-IFNγ, anti-IRF8, or anti-STAT4 using BD Cytofix/Cytoperm kit (BD Biosciences) for intracellular cytokine staining. Functional γδ T cell analysis was done by stimulation with BD leukocyte activation cocktail  for 4 hours prior to staining. Flow cytometry data were acquired using a BD LSRFortessa and analyzed by FlowJo software (BD Biosciences). Cell culture supernatant was analyzed for IL-12p70 using the BioLegend ELISA MAX Deluxe Set Mouse IL-12 (p70) kit per manufacturer instructions.MLN from Y. pseudotuberculosis strains were cultured overnight at 28°C and 220 RPM in LB media the night prior. The following morning, Y. pseudotuberculosis was sub-cultured 1:10 in LB and 50 mM CaCl2 at 37°C and 220 RPM for approximately 2 hours. Stimulation doses were based on the OD600. Cells were counted using a Vi-CELL Viability Analyzer (Beckman Coulter). Cells were stimulated in 96 round-bottom tissue culture treated plates with various strains of Y. pseudotuberculosis at 1 MOI at 37°C/5% CO2. 1x penicillin and streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin) were added to cells 2 hours post-stimulation. Cells were stimulated for a total of 24 hours or as indicated. BD GolgiPlug (BD Biosciences) was added 5 hours prior to the end of stimulation. If translocation was assessed, β-lactamase Loading Solutions kit (Invitrogen) was used to load CCF4-AM by incubating CCF4-AM at RT with cells for 1 hour in the dark. Cells were then processed for surface staining via incubation with live/dead stain, antibody, and Fc block (BioXcell) for 20 min in the dark at 4°C. Antibodies used included antibodies specific to Vδ2, CD3, TCRδ, (BioLegend). Cells were fixed, permeabilized, and stained with anti-IFNγ using BD Cytofix/Cytoperm kit (BD Biosciences) for intracellular cytokine staining. Flow cytometry data were acquired using a BD LSRFortessa and analyzed by FlowJo software (BD Biosciences).Blood was drawn and collected from healthy human donors in BD Vacutainer sodium heparin tubes (BD Biosciences). Blood was diluted 1:1 with 1x PBS at room temperature. Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coat of Ficoll-paque PLUS gradient centrifugation  for 20 min at 1,400 × g without a brake. PBMC were washed with 1x PBS at room temperature and resuspended in IMDM supplemented with 10% fetal bovine serum, 0.01 M HEPES, 100 μM Non-essential amino acids (Gibco), 2 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl or 1x Glutamax (Gibco), and 1 mM sodium pyruvate. After surface staining for flow cytometry, cells were washed and stained for pSTAT4 using a methanol-based approach. Cells were fixed in 4% PFA/1.5% methanol for 30 minutes in the dark at 4°C. Cells were then washed and incubated in methanol in the dark at 20°C for 45 minutes. After washing, cells were stained with anti-pSTAT4 (Y693)-PE (BD Biosciences) and washed once more. Flow cytometry data were acquired using a BD LSRFortessa and analyzed by FlowJo software (BD Biosciences).Y. pseudotuberculosis burden per organ was calculated.MLN were crushed and diluted in media prior to plating on LB agar. Total Y. pseudotuberculosis stimulation as described above. Cell preparations were stimulated with 10 MOI of WT or YopJC172AY. pseudotuberculosis or 1 MOI of WT Yptb-βla for 24 hours. 500 Vγ1.1/2- CD44hi CD27- γδ T cells were flow sorted directly into a tube with NEBNext Cell Lysis Buffer and Murine RNase Inhibitor and processed for RNA sequencing using NEBNext Single Cell/Low Input RNA Library Prep Kit (Illumina). Sequencing was performed at the Cold Spring Harbor Laboratory sequencing core on a NextSeq500. Fastq files were produced as an output of the sequencing files. Fastq were run through FastQC to perform quality control of transcripts prior to alignment. Fastq files were pair-ended aligned to GRCm38/mm10 by way of HISAT2 and output as .BAM files [Samples were prepared after AM files . Raw couAM files . DimensiAM files . To deteAM files . Cutoff AM files . Parametin vitro according to published protocols [L. monocytogenes infection of Balb/c mice. Red blood cells were lysed with red blood cell lysis buffer or ammonium chloride for 1 minute and cells from the MLN and spleen were combined. γδ T cells were enriched by negative selection using the following rat IgG primary antibodies from BioLegend: anti-CD4 (clone GK1.5), anti-CD8α (clone 53–6.7), anti-B220 (clone RA3-6B2), anti-MHC-II (clone M5/114.15.2), and anti-CD11b (clone M1/70). The MACS goat anti-rat IgG kit (Miltenyi Biotec) was used per manufacturer instructions with MACS LD columns and a QuadroMACS magnet. Enriched cells were cultured in 48-well plates coated overnight with 5 ug/ml anti-TCRδ (clone GL3). Cells were cultured in RPMI 1640 supplemented with 25 mM HEPES (Gibco), 1x glucose (Gibco), 10 g/ml folate (Sigma Aldrich), 1x sodium pyruvate (Gibco), 5x105 M 2 beta-mercaptoethanol (Sigma Aldrich), 1x Glutamax (Gibco), 1x penicillin-streptomycin (Gibco), and 10% FBS with 100 U/ml recombinant human IL-2. After 2 days of culture, cells were transferred into new wells with the same culture media to rest for 5 days. Cells were then stimulated with 10 MOI YopJC172AY. pseudotuberculosis with 0.1, 1, or 10 ng/ml of recombinant murine IL-12p70 (Peprotech).γδ T cells were expanded rotocols . The MLNC172AY. pseudotuberculosis infection and on day 2, 4, and 6 after infection, mice were treated with 0.2 mg of anti-IL-12p40  by i.p. injection. Blood was collected via tail vein on day 2, 4, and 6 after infection. Blood was incubated at ambient temperature for 30 minutes before being spun down at 1500G for 10 minutes at 4°C. Serum was isolated and analyzed for IL-12p70 with the BioLegend ELISA MAX Deluxe Set Mouse IL-12 (p70) kit.On the day of foodborne WT or YopJY. pseudotuberculosis stimulation of MLN cell suspensions, cultures were treated with 10 μg/ml anti-IL-12p40  for neutralization. In other conditions, recombinant murine IL-12p70 (Peprotech) was added at the start of Y. pseudotuberculosis stimulation of MLN cell suspensions at 2, 10, or 50 ng/ml.At the start of p < 0.05 was considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.GraphPad Prism 6 software (GraphPad Software Inc.) was used for statistical analysis. The differences between the means were compared using the statistical analysis described in the associated figure legends. All the data are presented as mean ± SEM and S1 Data(XLSX)Click here for additional data file.S1 FigA) MLN suspensions from L. monocytogenes infected mice were stimulated with WT or ΔYopB Y. pseudotuberculosis containing a β-lactamase translocation reporter (Yptb-βla) for 2 hours and given antibiotics. Cells were loaded with CCF4-AM dye to measure β-lactamase activity. FITC indicates CCF4-AM loaded cells without translocation (Yop-) and BV421 indicates CCF4-AM loaded cells with Yop translocation (Yop+). Vγ1.1/2- CD44hi CD27- γδ T cells were analyzed for Yop translocation 2 hours post stimulation at an MOI of 10. Representative contour plots are displayed. (B) MLN from L. monocytogenes infected mice were stimulated with Yptb-βla for 2 hours and given antibiotics. Yop translocation was detected as described above. The indicated cell populations were analyzed for Yop translocation 2 hours after stimulation. Representative contour plots are displayed. (C) MLN from L. monocytogenes infected mice were stimulated with Yptb-βla for 2 hours and given antibiotics. Vγ1.1/2- CD44hi CD27- γδ T cells were analyzed for Yop translocation 2 hours post stimulation at the indicated MOI and quantified for Yop translocation. Data consists of one experiment with 2–10 mice/group and the graphs depict the mean ± SEM in (A-C). ****p < 0.0001, ***p < 0.001, and **p < 0.01. A t-test was used for (A), and a repeated measures one-way ANOVA was used for (C). Comparisons were performed to ΔYopB Y. pseudotuberculosis in (A) and as depicted in (C).((TIF)Click here for additional data file.S2 FigA) MLN from L. monocytogenes infected mice were isolated and processed into single cell suspensions. Vγ1.1/2- CD44hi CD27- γδ T cells, CD4 T cells, and CD8 T cells were analyzed for β1-integrin expression. (B) MLN suspensions from L. monocytogenes infected mice were loaded with CCF4-AM dye and stimulated with 10 MOI WT Y. pseudotuberculosis containing a β-lactamase translocation reporter. CCF4-AM dye reports the occurrence of β-lactamase activity and Yop translocation. γδ T cells that contain Yop (Yop+) or do not contain Yop (Yop-) were analyzed for β1-integrin expression 2 hours after stimulation. Representative histogram plots are displayed. Data is pooled from two experiments with a total of 7 mice/group and the graphs depict the mean ± SEM in (A-C). ****p < 0.0001 and ***p < 0.001. A repeated measures one-way ANOVA was used for (A) and a t-test was used for (B), and. Comparisons were done to adaptive γδ T cells in (A) and as depicted in (B).((TIF)Click here for additional data file.S3 FigA and B) MLN from L. monocytogenes infected mice were stimulated with 10 MOI of WT Y. pseudotuberculosis (WT) or mutant YopJ Y. pseudotuberculosis (YopJC172A) for 24 hours. Antibiotics were given 2 hours post-stimulation. Five hundred Vγ1.1/2- CD44hi CD27- γδ T cells from each stimulation were flow sorted and processed for RNA sequencing. The heat map depicts upregulated genes in Vγ1.1/2- CD44hi CD27- γδ T cells after YopJC172AY. pseudotuberculosis stimulation and individual genes are listed. (C-E) Genes differentially expressed (downregulated) that overlapped between RNA sequencing analyses as displayed in the Venn diagram in (C) to select for direct effects of YopJ on Vγ1.1/2- CD44hi CD27- γδ T cells. The heat map depicts downregulated genes in Vγ1.1/2- CD44hi CD27- γδ T cells from the analysis in (C). Individual genes are listed in (E). Each experiment was performed once with biologic replicates. The cutoff for gene significance was p < 0.05 and FDR < 0.10.((TIF)Click here for additional data file.S4 FigL. monocytogenes infected mice were stimulated with 10 MOI of WT, YopJC172A, or ΔYopB Y. pseudotuberculosis for 24 hours. Antibiotics were given 2 hours after stimulation and brefeldin A was added for the last 5–6 hours. Vγ1.1/2- CD44hi CD27- γδ T cells were analyzed for IL-17A production after stimulation. Representative histograms are displayed. The graph depicts mean ± SEM and represents two independent experiments with 4 mice per group. A repeated measures one-way ANOVA was used. * p < 0.05.MLN cell suspensions from (TIF)Click here for additional data file.S5 FigA) Homer motif analysis was performed on the RNA sequencing results for the YopJC172A and WT Y. pseudotuberculosis comparison from C172A stimulated Vγ1.1/2- CD44hi CD27- γδ T cells. (B) MLN from L. monocytogenes infected mice were stimulated with 10 MOI of WT, YopJC172A, or ΔYopB Y. pseudotuberculosis for 6 hours. Antibiotics were given 2 hours post-stimulation. Vγ1.1/2- CD44hi CD27- γδ T cells were analyzed for IRF8 levels with or without anti-IL12p40 neutralizing antibody. The graph depicts the percentage of IRF8 protein expression among Vγ1.1/2- CD44hi CD27- γδ T cells after WT, YopJC172A, or ΔYopB Y. pseudotuberculosis stimulation. Data depict two pooled experiments with a total of 8 mice/group and represents the mean ± SEM. (C) The genes from the RNAseq and Homer motif analysis in Figs D) STAT4 KO spleens are shown in maroon and WT spleens are shown in black in representative histograms. The graph depicts the MFI of STAT4 protein expression in bulk γδ T cells. Data depicts one experiment with 4 mice/group and represents the mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. A repeated measures one-way ANOVA was used for (B), and a t-test was used for (D). Comparisons were performed as depicted in (B) and to Naïve WT in (D).((TIF)Click here for additional data file.S6 FigA) γδ T cells enriched from the MLN and spleen of L. monocytogenes infected mice were expanded with plate bound γδTCR antibody for 2 days and rested for 5 days. After expansion, ~ 50% of cells were γδ T cells, and the majority of those were Vγ4 T cells. The enrichment summary reflects the mean enrichment from 4 samples. Afterwards, γδ T cells were isolated from cultures and stimulated with YopJC172AY. pseudotuberculosis with 0.1, 1, or 10 ng/ml IL-12p70 for 24 hours. Antibiotics were added 2 hours after stimulation and brefeldin A was added for the last 5–6 hours. Histograms display IFNγ production from Vγ1.1/2- CD44hi CD27- γδ T cells under different culture conditions. Data depicts one experiment with 4 mice pooled and split into the indicated stimulation conditions. (B) MLN cell suspensions from L. monocytogenes infected mice were stimulated with 10 MOI of WT Y. pseudotuberculosis in the presence of 2, 10, or 50 ng/ml IL-12p70 or 10 MOI of YopJC172AY. pseudotuberculosis for 24 hours. Antibiotics were given 2 hours after stimulation and brefeldin A was added for the last 5–6 hours. Vγ1.1/2- CD44hi CD27- γδ T cells were analyzed for IFNγ production. Representative histograms of IFNγ production from Vγ1.1/2- CD44hi CD27- γδ T cells are displayed. The graph depicts mean ± SEM from one experiment with 4 mice per group *p < 0.05. A repeated measures one-way ANOVA was used for comparisons to YopJC172AY. pseudotuberculosis in (B).((TIF)Click here for additional data file.S7 FigA) Balb/c mice were foodborne infected with the indicated doses of WT or mutant YopJC172AY. pseudotuberculosis and tissues were analyzed 9 days post-infection. Bacteria burden was quantified from the MLN. Data reflect 3–5 mice per group pooled from 3 independent experiments and the graphs depict the mean ± SEM. (B) Balb/c mice were foodborne infected with the indicated doses of WT or mutant YopJC172AY. pseudotuberculosis. Nine days post infection, MLN suspensions from WT or YopJC172AY. pseudotuberculosis infected mice were stimulated with PMA/ionomycin and brefeldin A for 4 hours. Vγ1.1/2- CD44hi CD27- γδ T cells were analyzed for IFNγ production. Representative histograms are displayed and quantified. Data depicts one experiment with 3 mice per group. (C) Balb/c mice were foodborne infected with WT (2-4x107 CFU) or YopJC172AY. pseudotuberculosis (2-4x108 CFU) and treated with 0.2 mg/mouse of anti-IL12p40 on days 0, 2, 4, and 6 post infection. IL-12p70 concentrations were determined from serum at days 2, 4, and 6 post infection. Data represent 2 independent experiments with a total of 9 mice per group. Serum samples were pooled into groups of 3 per experimental condition. (D) Balb/c mice were foodborne infected with 2x109 CFU L. monocytogenes to elicit a Vγ1.1/2- CD44hi CD27- γδ T cell population in vivo. 30 days post infection, adaptive Vγ1.1/2- CD44hi CD27- γδ T cells from the MLN of immune mice were analyzed for Yop translocation using the CCF4-AM assay. Representative contour plots are shown. Yop translocation (Yop+) among the indicated populations represents background staining as a negative control for A), and an unpaired t-test was used for (B). Comparisons were performed to uninfected in (A) and to 5x107 WT Y. pseudotuberculosis in (B).((TIF)Click here for additional data file.


Scientific Reportshttps://doi.org/10.1038/s41598-021-00232-7, published online 29 October 2021Correction to: The original version of this Article contained errors.In Figure 2e , a grey data point located above the error bar in the “Control” bar was omitted. Additionally, in Figure 2f , a grey data point located above the error bar in the “Co-cultured with M2-like” bar was omitted.+”) the y-axis label “0” and the grey data point located above the bar “Co-cultured with M1” were duplicated.Furthermore, in Figure 3C (a) (middle graph, “Day7 CD206The original Figure The original Article has been corrected.