The catalysts were characterized by XRF, XRD, textural properties, XPS, Raman, Py-TPD, and EDS elemental mapping. The chemical analyses by X-ray fluorescence (XRF), EDS elemental mapping, and CHNS-O elemental analysis showed stoichiometric values Al/Mg = 38–40, Mo:Ni:P ∼ 1, and S ≤ 4.5 wt % ; also EDS elemental mapping confirmed the presence of Mo, Ni, Al, O, P, Mg, and S with good distribution on Al2O3-MgO. The impregnation of metals leads to a decrease in the surface area and pore volume as follows NiMoP-S/γ-Al2O3-MgO < MoP-S/γ-Al2O3-MgO < NiP-S/γ-Al2O3-MgO < Al2O3-MgO < Al2O3 , propitiating a pseudo bimodal pore size distribution with Dp-BJH between ∼5–7 nm and 11.8–14.2 nm for the presence of MgO. XRD confirmed differences between metallic phosphates and phosphides, and XPS confirmed the presence at the surface of Moδ+(0 < δ+ < 2), Mo4+, Mo6+, Niδ+(0 < δ+ < 2), Ni2+, S2−, SO42−, Pδ+, and P5+ species. Raman revealed the presence of MoS2 only in MoP-S/γ-Al2O3-MgO and NiMoP-S/γ-Al2O3-MgO, while the NiMoP-S/γ-Al2O3-MgO catalyst had a more significant number of Brønsted and Lewis sites, although the increasing temperature decreased the Lewis sites. MoP-S/γ-Al2O3-MgO was more active at HDO showing the highest production rate for octadecane of 53 mol/(gcat·h), whereas NiP-S/γ-Al2O3-MgO was more active at HDCx-HDCn [45 mol/(gcat·h)] and HCK [6 mol/(gcat·h)]; meanwhile, NiMoP-S/γ-Al2O3-MgO had a mix of HDO [47 mol/(gcat·h)] and HDCx-HDCn [41 mol/(gcat·h)]. This showed production towards octadecane, heptadecane, and light hydrocarbons, meaning that the fatty acids were deoxygenated through bifunctional sites for hydrogenation (HYD) and hydrogenolysis (HYG) as follows: MoP-S/γ-Al2O3-MgO (K1 = 0.08 and K2 = 0.03 L/mol) < NiMoP-S/γ-Al2O3-MgO (K1 = 0.25 and K2 = 0.45 L/mol) < NiP-S/γ-Al2O3-MgO (K1 = 2.5 and K2 = 6.5 L/mol). For this reason, we considered that phosphide acts as a structural promoter with sulfur on its surface as a “mixed phosphidic-sulphidic species”, allowing the largest generation of heptadecane and octadecane by the presence of BRIM sites for HYD and CUS sites for HYG.The effect of the sulfur and metal-type content of MoP-S/ γ-Al2O3-MgO, NiMoP-S/γ-Al2O3-MgO, and NiP-S/γ-Al2O3-MgO influence the reaction pathways for hydrotreating fatty acids, improving the dispersion of MoS2 on phosphide “mixed phosphidic-sulphidic species” and increasing the number of Lewis and Brönsted sites, which correlates well with the HDO and HDCx-HDCn pathways.The sulfur and metal-type content in MoP-S/ Thus, the increased consumption of oil derivatives quickly reduces world crude oil reserves . Hence, A fact that has been gaining attention in recent years is the use of biofuels that have compositions resembling that of fossil-derived fuel and that avoid the presence of glycerol (petro-diesel), which is called “renewable diesel” . This reIn general, the hydrotreating of triglycerides and fatty acids is the removal of oxygen yielding high-quality diesel fuel by three reaction pathways: decarboxylation (DCx), decarbonylation (DCn), and hydrodeoxygenation (HDO) . Three cThe nature of the support can influence the product distribution of the HDO reaction . The mos2O3 for HDO of pyrolysis oil, reducing the oxygen content from 41.8 wt % to 3 wt .%, which suggests that phosphorus promotes the activation of hydrogen in the hydrotreatment process. Hence, transition metal phosphide catalysts have been studied extensively because they are economical and less susceptible to hydrogenolysis of the C-C bond  because they have shown excellent HDO performance P/MCM-41 > NiMo(1:1)P/MCM-41 ∼ (Ni2P + MoP)/MCM-41 > NiMo(1:3)P/MCM-41 > MoP/MCM-41, suggesting that the Ni was the main active site, while Mo determined the selectivity. Although sulfided MgO-supported catalysts have been the object of a limited number of research studies 2]3, 99.8%, Sigma- Aldrich} was dispersed in 50 ml of isopropanol  under magnetic stirring at about 343 K until a homogeneous solution was obtained.The Alynthesis . In a ty3)2·6H2O, 98%, Sigma-Aldrich] was added to the solution of Al[OCH(CH3)2]3/(CH3)2CHOH at 350 K for 4 h and then cooled to room temperature until the polymerized solution of Mg(NO3)2·6H2O/Al[OCH(CH3)2]3 in alcoholic medium was obtained. The solution was then slowly added to a surfactants solution that was obtained by homogenizing 56.5 mmol of tetramethylammonium hydroxide  in 30 ml of deionized water followed by the addition of 18.1 mmol of hexadecyltrimethylammonium bromide  until complete dispersion.An aqueous solution of magnesium nitrate hexahydrate  and/or ammonium heptamolybdate  with ammonium dihydrogen phosphate  in stoichiometric amounts of Mo:Ni:P = 1:1:1 (20 wt.% Mo) to a flask containing the support, under stirring at 343 K. The impregnation step lasted until removal of the solvent by evaporation. Finally, the mass obtained was further dried at 373 K for 12 h and the sample was calcined at 823 K for 4 h. The precursors were transferred to a fixed-bed tubular stainless-steel reactor  with an axial thermowell containing a thermocouple centered in the catalyst bed deposited on a quartz fiber bed. A tubular electrical furnace controlled by a temperature programmer  was used to heat the reactor. The amount of the sample was approximately 2 g/batch. Pure H2 was passed through the sample at a total flow rate of 50 cm3 min−1. The temperature was increased at a linear rate of 1°C min−1 to the final temperature (973 K), which was held for 1 h  calculated the surface areas of samples, and total pore volume and pore size distribution were determined from the desorption branch of the isotherm using the Barret–Joyner–Halenda (BJH) model (micro) was obtained from a mass balance, i.e., Smicro = SBET − Sext (4)3PO4(MoO3)12•4H2O (card no. 09-0408), MoP (card no. 24-0771), NiMoP (card no. 031-0873), and γ-Al2O3 (card no. 10-0425). Raman spectra of the phosphides were obtained in a Horiba Scientific Raman microspectrometer with LabRAM HR Evolution at room temperature using the 532 nm line and a between 41.1 and 56.1 μW He–Ne laser excitation source through an Olympus TM BX41 optical microscope. FTIR measured the pyridine adsorption on the different samples with the aim to monitor both the type (Brønsted or Lewis acidic sites) and their relative abundance (−2) and placed into a special IR cell; then, the sample was immediately pretreated under vacuum at 773 K for 4 h. Afterwards, the sample temperature was decreased to 423 K and exposed to pyridine vapor (1.5 m bar pressure) for 5 min. The physically adsorbed pyridine fraction was removed by degassing the sample for 30 min, and the IR spectra were collected. Spectra were recorded in 256 scans using a Nicolet FTIR spectrometer, and the data analysis was performed with the spectrometer software Omnic 8.1. The adsorbed pyridine on Brönsted and Lewis acid sites was calculated under different desorption temperatures . The acidity due to Brönsted and Lewis sites , in μmol Py/g, was calculated as:−1 in the absorption spectrum of pyridine, IMECB and IMECL are the integrated molar extinction coefficients corresponding to the bands at 1,450 and 1,540 cm−1, IMECL = 222 cm/μmol and IMECB = 167 cm/μmol (solid is the mass of the sample (g).Elemental analysis for Mo, Ni, P, S, and Mg was determined by X-ray fluorescence using a MagixPro PW–2440 Philips instrument. The textural characteristics of the catalysts were determined employing the physisorption of NH) model . The totT − Sext . XRD anaT − Sext , the JCPbundance . A thin  cm/μmol , and mso–9 torr. Calibration of the instrument was carried out employing the Au 4f7/2 line at 83.9 eV. A dual flood gun source of low-energy electrons and Ar+ ions was used during all measurements to prevent surface charging. The internal referencing of binding energies was made using the dominating Al 2p band of the support at 74.4 eV. After in situ sulfiding for XPS analysis, samples were immersed in purified n-hexane and quickly transferred to the XPS instrument, where the solvent was slowly removed under vacuum (The sulfided phosphides’ surface composition was determined through X-ray photoelectron spectroscopy (XPS) with a Thermo Scientific K-Alpha spectrometer, equipped with a dual (non-monochromatic) Mg/Al anode, operated at 400 W and under a vacuum better than 10r vacuum . Curve fScanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX) analysis were undertaken using a field emission scanning electron microscope  operated at 1 kV. The EDX chemical elemental mapping images and elemental analysis were acquired simultaneously at 15 kV using the energy-dispersive X-ray spectroscopy interconnected with the same JEOL 7800F instrument.Sulfur elemental analysis of the mixed phosphidic-sulphidic phase was carried out using a combustion method using a Fisons EA 1108 CHNS-O analyzer.in situ under a 0.5 vol % CS2/heptane mixture and H2 (50 cm3 min−1) at 300°C for 1 h. The hydrotreatment of the mix of fatty acids (oleic and stearic acids) was conducted under conventional hydrotreating conditions in a 600 ml batch reactor (2 at ambient temperature and then pressured to 3.1 MPa H2. The reaction mixture was then heated from room temperature to reaction temperature (573 K) and target H2 pressure (5 MPa H2) for 6 h under constant stirring (300 rpm). The consumption during the reaction of fatty acids and the products contained in the liquid phase were collected and injected into a CG-2014 Shimadzu equipped with a flame ionization detector (FID). The products were identified and confirmed by GC-MS Agilent technology 7890B GC System-5977A MSD and compared to standards. The effluents sampling of the reactor occurred at 0, 90, 180, 270, and 360 min, and the catalytic activities of the catalysts were determined (−1) at the beginning and at 6 h, respectively.To remove the passivation layer and attain a stable and reproducible Ni-Mo mixed phosphidic-sulphidic phase , before  reactor . In a tytermined . The con−1) at the beginning and product formed at 6 h, respectively.Additionally, the yield of fatty acids HYD was calculated as the proportion of a product formed in relation to the fatty acids consumed during the reaction as defined in Both the supporting alumina and the Mg-modified alumina showed negligible fatty acids conversion in the reaction. Established procedures verified the absence of mass and heat flow transport effects . All expAll the reactions were assumed irreversible due to the excess of hydrogen (considered constant), whereby the reactions were considered pseudo-first-order. The semi-empirical kinetic model of fatty acids HYD is the generalized Hinshelwood-Hougen-Watson model . It was In 1, k2, k3, k4, k5, k6, and k7 and K1 and K2, respectively.The system of differential equations to calculate the apparent rate and equilibrium adsorption constants was solved using the Mathematics-Wolfram software version 11.1. The nonlinear parameter estimations were calculated until converged by minimizing the deviation from theoretical and experimental concentrations (objective function) at different times using the Levenberg–Marquardt algorithm of GNU Octave version 4.2.2 through the Optim package, with the constraint of non-negativity. In this approach, the apparent rate and equilibrium adsorption constants of the oleic and stearic acids HYD were calculated as kcat, g) of the catalyst after 6 h of reaction time. Therefore, the HDO, DCx-DCn, and hydrocracking (HCK) activities were reported after 6 h of reaction time as defined in Finally, the fatty acids HYD activities of the catalysts were reported as pseudo-first-order rate constant for oleic and stearic acid disappearance normalized by the moles of oleic or stearic acids per weight , which does not show characteristic signs of a metal phosphate or phosphide. It is worth mentioning that it is difficult to distinguish the diffraction peaks, referring to phosphide/sulfide and MgO (∼2 wt%) in all the catalysts, probably because alumina amorphism, crystallites size  or the particles of phosphidic-sulphidic composite well dispersed on the γ-Al2O3-MgO matrix. However, due to the changes in the difractograms between metallic phosphates and phosphides, it lets assume that under the reaction conditions it is likely to generate these types of species, such as MoP at 2θ = 32.17 and γ-Al2O3 at 2θ = 67.10, 45.90, 37.64 in MoP-S/γ-Al2O3-MgO (E and F patterns); and NiMoP at 2θ = 43.19; (NH4)3PO4(MoO3)12•4H2O at 2θ = 26.47, 21.51 and γ-Al2O3 for NiMoP-S/γ-Al2O3-MgO as will be displayed in XPS (G and H patterns).The γ-Al2O3-MgO support. The NiP-O/γ-Al2O3-MgO precursor presented the main band at approximately 1,000 cm−1, characteristic of antisymmetric stretching modes of P-O bonds (PO43− stretching) , 960, 922, 830 (shoulder), 588, 382, and 226 cm−1 (shoulder) that could be assigned to the PO34− group, MoO2t symmetric stretching, MoO42−, Mo–O–Mo bridge stretching, stretching of Mo–O–Al entities, and terminal Mo=O of octahedral MoO42− species, respectively , 952, 906, 838, 360–390, and 224 cm−1 that could be assigned to the PO34− group, MoO2t symmetric stretching, MoO42−, stretching of Mo–O–Al or Mo–O–Ni entities, and terminal Mo=O of octahedral MoO42− species, respectively  , while Nng modes . MoP-O/γectively . NiMoP-Oectively . The Ramred MoS2 . We suppred MoS2 . The ban2 physisorption isotherms of Al2O3, Al2O3-MgO, NiP-S/γ-Al2O3-MgO, MoP-S/γ-Al2O3-MgO, and NiMoP-S/γ-Al2O3-MgO were type IV in the IUPAC classification  > r Al3+ (0.051 nm)] due to the sol-gel method that forms Al-O-Mg bonding in the process of gelation  , with vation see   of nickel phosphide, Ni2+ ions interacting with phosphate or sulfates, and the strong shake-up lines characteristic of Ni2+ species in an Ni-O-P/Ni-O-S matrix, respectively. Note that the electron binding energy of Niδ+ was more extensive than that of metallic Ni, suggesting the charge transfer from Ni to P , Mo4+ (Mo 3d5/2 = 229 eV), and Mo6+ (Mo 3d5/2 = 232.5 eV) species due to Mo-P, MoS2, and MoO3, respectively (δ+ and Mo4+ species (sulfided). Additionally, in the Mo 3d5/2-3/2 region, a shoulder at 226.5 eV was found, corresponding to the 2 s signal of sulfur, which can be confirmed by the presence of two bands in the S 2p3/2 region (2−) ligands in MoS2 or NiMoS phases and the signal at 168.9 eV, which can be assigned to SO42− , 523 K (BS = 15.2 μmol/g and LS = 89.8 μmol/g), and 623 K (BS = 15.7 μmol/g and LS = 36.9 μmol/g). However, as the temperature increases, it is observed that the amount of the Lewis sites in NiMoP-S/γ-Al2O3-MgO was much less than that of NiP-S/γ-Al2O3-MgO , suggesting that these sites in the NiP-S/γ-Al2O3-MgO catalyst are stronger that NiMoP-S/γ-Al2O3-MgO; moreover, their Brönsted acid sites did not produce appreciable changes  as seen from 3) for catalyst NiP-S/γ-Al2O3-MgO was much lower than that for other catalysts, which must be related to the oxophilic capacity of molybdenum (1+k4 (HDO), k5 (HDCx-HDCn) and k6+k7 (HCK)] of γ-Al2O3-MgO was more active at HDO, showing the highest production rate for octadecane of 53 mol/(gcat·h), whereas NiP-S/γ-Al2O3-MgO was more active at HDCx-HDCn [45 mol/(gcat·h)] and HCK [6 mol/(gcat·h)], and NiMoP-S/γ-Al2O3-MgO had a mix of HDO [47 mol/(gcat·h)] and HDCx-HDCn [41 mol/(gcat·h)], which showed production towards octadecane, heptadecane, and light hydrocarbons (γ-Al2O3-MgO and NiMoP-S/γ-Al2O3-MgO showed the highest HYD from oleic acid to stearic acid of 16.2 and 12.4 mol/(gcat·h), respectively. Therefore, the previous results confirm that MoP-S/γ-Al2O3-MgO and NiMoP-S/γ-Al2O3-MgO catalysts had a higher percentage of the HDO route . Although the surface area and pore size (similar) did not directly correlate with the catalytic activity, the decreasing pore volume probably allows greater exposure of the active sites to the catalysis regardless of which route is taken. In this regard, the HDO, HDCx-HDCn, and HCK activities can be correlated with the number and nature of the active sites on the surface (Lewis and Brönsted sites).The fatty acids HYD activities function of the product conversion of the NiP-S/lybdenum . Hence, lybdenum . The pseocarbons . LikewisDO route ; while Nδ+/Mo4+  on the surface combined with Brönsted and Lewis acidity of the material that preferentially increases the rate of C-O bond cleavage reactions, i.e., NiMoP-S/γ-Al2O3-MgO and MoP-S/γ-Al2O3-MgO have a lot of Brönsted acid sites compared to NiP-S/γ-Al2O3-MgO. Thus, the molar ratio between the HDO and HDCx-HDCn (denoted as (k4+k1)/k5 ratio) can represent the selectivity between the decarbonylation and HDO pathways ] adsorb the oxygen favoring the activation and dissociation of C=O and C-O bonds by Brønsted acid sites, i.e., neighboring SH and/or OH groups as mentioned by Wang ; this is probably due to the strength of the acid site and not to the number of Niδ+ species. Chen and Ruinart reported that Ni-based catalysts such as Ni, Ni2P, and Ni3S2 promote the decarbonylation/decarboxylation pathway  would be an accurate representation for the active phase in typical hydrotreating conditions in these materials ; otherwise it is observed in MoP-S/γ-Alreaction . We consγ-Al2O3-MgO, NiMoP-S/γ-Al2O3-MgO, and NiP-S/γ-Al2O3-MgO on hydrogenation of fatty acids was evaluated. The chemical analyses by FRX for MoP-S/γ-Al2O3-MgO, NiMoP-S/γ-Al2O3-MgO, and NiP-S/γ-Al2O3-MgO showed stoichiometric values for Al:Mg, Ni:Mo, Ni:P, and Mo:Ni:P of 38 and ∼1, while EDS spectra and elemental mapping confirmed the presence of Mo, Ni, Al, O, P, Mg, and S with good distribution on Al2O3-MgO. The XRD patterns revealed differences between metallic phosphates and the phosphidic-sulphidic phase. However, it was difficult to distinguish a phosphide phase because the crystallite size was too small, and the phosphide particles were well dispersed on the support. MoP-S/γ-Al2O3-MgO and NiMoP-S/γ-Al2O3-MgO showed two typical Raman peaks at 380 and 405 cm−1 for the E12g and A1g active modes characteristic of the bulk of multilayered MoS2. All the N2 physisorption isotherms of the materials were type IV with H1 and H2 hysteresis loops. MoP-S/γ-Al2O3-MgO and NiP-S/γ-Al2O3-MgO showed better textural properties than NiMoP-S/γ-Al2O3-MgO with an average pore radius between 6.5 and 11.2 nm. XPS evidenced Moδ+(0 < δ+ < 2), Mo4+, Mo6+, Niδ+(0 < δ+ < 2), Ni2+, Pδ+, and P5+ species, which showed a dependence with the type of metal and the presence of sulfur. The FTIR Py analysis of MoP-S/γ-Al2O3-MgO, NiMoP-S/γ-Al2O3-MgO, and NiP-S/γ-Al2O3-MgO showed the main band characteristics of the pyridine adsorbed on Lewis acid sites and much lower amounts of Brønsted acid sites. The NiMoP-S/γ-Al2O3-MgO catalyst had a more significant number of Brønsted and Lewis sites (15.7–34.2 μmol/g vs. 36.9–106.6 μmol/g). However, the Lewis sites in NiP-S/γ-Al2O3-MgO were stronger that NiMoP-S/γ-Al2O3-MgO. MoP-S/γ-Al2O3-MgO was more active at HDO showing the highest production rate for octadecane of 53 mol/(gcat·h), whereas NiP-S/γ-Al2O3-MgO was more active at HDCx-HDCn [45 mol/(gcat·h)] and HCK [6 mol/(gcat·h)], and NiMoP-S/γ-Al2O3-MgO had a mix of HDO [47 mol/(gcat·h)] and HDCx-HDCn [41 mol/(gcat·h)]. The higher HDO and HDCx-HDCn activities could be ascribed to the high hydrogenolysis capacity of molybdenum sulfide . The (k4+k1)/k5 ratios were between 0.19 and 1.81 on all catalysts at 300°C, meaning that the oleic and stearic acids were deoxygenated mainly through the HDO pathway as follows: NiP-S/γ-Al2O3-MgO < NiMoP-S/γ-Al2O3-MgO < MoP-S/γ-Al2O3-MgO. The catalysts had bifunctional sites, leading to an increase of the adsorption constants as follows: MoP-S/γ-Al2O3-MgO (K1 = 0.08 and K2 = 0.03 L/mol) < NiMoP-S/γ-Al2O3-MgO (K1 = 0.25 and K2 = 0.45 L/mol) < NiP-S/γ-Al2O3-MgO (K1 = 2.5 and K2 = 6.5 L/mol). Hence the high production of heptadecane and octadecane by the presence of mixed NiMo phosphidic-sulphidic species (BRIM sites for HYD and CUS sites for HYG) suggests a type of synergism between the Lewis and Brönsted sites of the catalyst giving an optimum “bond strength” defining the best catalyst for a given reaction.The effect of phosphidic-sulphidic phase content in MoP-S/


N-{3-cyano-1--4-(ethylsulfanyl)-1H-pyrazol-5-yl}-2,2,2- trifluoroacetamide (C15H8N4Cl2F6OS) are presented.The synthesis, crystal structure, and some spectroscopic details for the phenylpyrazole-based insecticide  15H8N4Cl2F6OS, a phenylpyrazole-based insecticide related to ethiprole, fipronil, and derivatives thereof is presented. The pyrazole ring has four chemically diverse substituents, namely a nitrogen-bound 2,6-dichloro-4-trifluoromethylphenyl and carbon-bound cyano, ethylsulfanyl, and 2,2,2-trifluoroacetamide groups. The pyrazole and phenyl rings are perpendicular, subtending a dihedral angle of 89.80 (5)°. In the crystal, strong N—H⋯O hydrogen bonds link the molecules into chains that extend parallel to the a-axis.The structure of the title compound, C Further addition of any sulfur-bound substituent at the equivalent of C2 gave nine hits, only eight of which are unique. Two of these structures, FOCCUW , triethylamine  and DCM (5 ml) at 273 K. The reaction was kept at 273 K for 5 h, warmed to room temperature over 3 h, quenched with water and extracted with DCM three times. An overall scheme for the reaction is shown in Fig. 6N-{3-cyano-1--4-(ethylsulfanyl)-1H-pyrazol-5-yl}-2,2,2-trifluoroacetamide .Trifluoroacetic anhydride  was added dropwise to a stirred solution of 5-amino-1--4-ethylsulfanyl-1The product was dissolved in ethanol at 333 K and stirred for 30 min. The resulting solution was allowed to cool slowly to room temperature with slow evaporation. X-ray-quality crystals appeared in two days (m.p. 366–367 K).1H NMR spectroscopies, as follows: FT–IR (ν in cm−1): 3227 (N—H stretching), 2250 (C=N stretching), 1737 (C=O stretching), 1694–1652 (C=C stretching), 1313, 1222 (C—F stretching), 881, 818 , 711, 628 (C—Cl). 1H NMR: DMSO–d6 : 12.42 , 8.36 , 2.90–2.85 , 1.19–1.15 .The title compound was characterized by IR and 6.RCH3), 0.99 Å (R2CH2) and 0.95 Å (R2CH). The nitrogen-bound hydrogen-atom coordinates were refined freely. Uiso(H) parameters were set to values of either 1.2Ueq or 1.5Ueq (RCH3 only) of the attached atom.Crystal data, data collection, and structure refinement details are summarized in Table 410.1107/S2056989022009653/tx2059sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989022009653/tx2059Isup2.hklStructure factors: contains datablock(s) I. DOI: 2210523CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Dietary supplements sold for anabolic benefits or performance enhancement often contain substances, which are non-approved and might lack quality controls. With regard to athletes, the inclusion of substances or methods in the prohibited list of the World Anti-Doping Agency is based on medical or scientific evidence. 5α-hydroxy-laxogenin is a synthetic spirostane-type steroid, which is contained in dietary supplements and advertised as anabolic agent. To date, evidence is missing on anabolic or androgenic activity of 5α-hydroxy-laxogenin. We investigated its androgenic potential in two in vitro bioassays. While no activity was observed in the yeast androgen screen, 5α-hydroxy-laxogenin was able to trans-activate the androgen receptor in human prostate cells in a dose-dependent manner. Interestingly, a biphasic response was observed with antagonistic properties at lower concentrations and agonistic effects at higher concentrations tested. The demonstrated androgenic properties of the higher concentrations demonstrate that further investigations should focus on the safety as well as on potential anabolic effects of 5α-hydroxy-laxogenin. This is of interest with regard to abuse for doping purposes. The World Anti-Doping Agency (WADA) publishes annually the lists of substances and methods prohibited as doping .Sigma-Aldrich  provided dihydrotestosterone , bicalutamide (purity ≥ 98%) and hydroxyflutamide . 5α-hydroxy-laxogenin  was purchased from Biomol . Carl Roth  provided dimethyl sulfoxide . All test compounds were dissolved in DMSO. Chlorophenol red-β-D-galactopyranoside was purchased by Roche . Dulbecco's Modified Eagle’s Medium (DMEM/F12), fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were supplied by BioWest . Qiagen  provided Attractene Transfection Reagent. G418 was supplied by Calbiochem, Merck . Luciferase assay system was supplied by Promega . AppliChem  provided bovine serum albumin fraction V (BSA). Dr. Aria Baniahmad  kindly provided human PC3(AR)Saccharomyces cerevisiae strain stably transfected with a human androgen receptor (AR) construct and a reporter plasmid carrying the β-galactosidase encoding LacZ gene under the control of androgen responsive elements. Yeast cells were either treated with DMSO as solvent control, DHT at concentrations of 10–11 M to 10–6 M as positive control or serial dilutions of 5α-OH-laxogenin in the range from 0.01 µg/mL to 100 µg/mL (2.2 × 10–8 M to 2.2 × 10–4 M respectively). For antagonization, 5α-OH-laxogenin was co-incubated with 10–5 M OH-flutamide. β-galactosidase activity was measured by hydrolysis of chloro-phenol red-β-D-galactopyranoside at 565 nm and corrected by the optical density at 690 nm respectively. All treatments were performed in three independent experiments. Statistical significance was assessed by Student’s t test considering p < 0.05 as significant.Yeast androgen screen was performed using a 2 cells were maintained in DMEM/F12 supplemented with 1% P/S, 0.25 mg/mL G418 and 10% FBS. For experiments, PC3(AR)2 cells were cultivated in DMEM/F12 containing 1% P/S and 5% dextran-coated charcoal-treated FBS. 70,000 cells seeded per well in a 24-well plate were transiently transfected with the reporter plasmid mmTV-luc (0.2 µg plasmid DNA and 0.5 µL Attractene per well). Cells were either treated with 0.1% DMSO as solvent control, 5 × 10–9 M DHT as positive control or 5α-OH-laxogenin, respectively . For antagonization experiments, cells were co-incubated with 5 × 10–7 M bicalutamide and 50 µg/mL 5α-OH-laxogenin. According to the manufacturer’s protocol, we measured luciferase activity and quantified protein concentration using the bicinchoninic acid assay with BSA as standard protein. We calculated Relative Luminescence Units (RLU) by normalizing the luminescence with the protein concentration. Three independent cell culture experiments were performed from which means ± standard deviation are shown. Statistical significance was assessed by one-way ANOVA followed by Bonferroni’s post hoc test, p < 0.05 as significant.Human PC3(AR)In the yeast androgen screen, DHT stimulated the reporter gene expression in a dose-dependent manner Fig. a. Howeve2 cells in a biphasic dose-dependent manner, with antagonistic effects at lower doses (0.01–1 µg/mL) and agonistic effects at higher doses The observed androgenic potential of higher 5α-hydroxy-laxogenin concentrations observed herein raises possible safety concerns regarding reproductive organs . Besides the androgenic activity, further investigations should focus on potential anabolic activities of 5α-hydroxy-laxogenin. The proof of anabolic effects would meet WADA’s criterion on performance enhancement. Moreover, data on pharmacokinetics as well as on biotransformation should be gathered to contribute to an estimation of the efficacy and safety of 5α-hydroxy-laxogenin.In conclusion, we showed androgenic potential of 5α-hydroxy-laxogenin in an in vitro bioassay for the first time. As this synthetic spirostane-type steroid is marketed as dietary supplement for athletes, future investigations focusing on potential anabolic properties and safety of 5α-hydroxy-laxogenin intake are necessary.


The methylphenyl ring is oriented at an angle of 36.2 (1)° with respect to the best plane of cyclohexane moiety. In the crystal, molecules associate via C—H⋯N hydrogen bonds, forming a three-dimensional network.In the title compound, C Atoms C1 and C4 deviate by 0.681 (1) and −0.652 (1) Å, respectively, from the least-squares plane through the remaining four atoms. An intramolecular C—H⋯O hydrogen bond is observed (Table 1S(6) ring ° with respect to the best plane of cyclohexane moiety. The cyclohexane ring adopts a chair conformation, the puckering parameters octahydronaphthalene-1,3,3(2H)-tricarbonitrile (0.01 mol), formic acid (5 mL) and a catalytic amount of concentrated HCl was refluxed for 16 h and the reaction mixture was allowed to cool. The reaction mixture was poured onto crushed ice and the solid that separated was filtered, dried and recrystallized using ethanol and water as mixed solvents.A mixture of 2-amino-4- I, shelx. DOI: 10.1107/S2414314621005009/bt4111Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314621005009/bt4111Isup3.cmlSupporting information file. DOI: 2057162CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound comprises a cobalt(II) atom, which is six coordinate by N donor atoms, four from isothiocyanato ligands and two from 1-dodecyl-4-aza-1-azoniabicyclo[2.2.2]octane cations. The coordination sphere is elongated octahedral. 18H37N2)2(NCS)4], consists of a cobalt(II) ion positioned on the origin of the triclinic unit cell. It is coordinated by the N atoms of two trans-oriented 1-dodecyl-4-aza-1-azoniabicyclo[2.2.2]octane (DABCO+) cations, which carry n-dodecyl chains at the non-coordinating N atoms. The distorted octahedral coordination environment of the CoII ion is completed through four N atoms of isothiocyanate ions, which are arranged within the equatorial plane. Non-classical hydrogen bonding of the types C—H⋯N and C—H⋯S between the filamentous molecules lead to the formation of layers parallel to (001).The title compound, [Co(C The N1—C1 distance measures 1.162 (3), Å indicating a strong π-interaction, and the C1—S1 distance is 1.629 (2) Å. The coordination polyhedron around the CoII ion consists of the four N atoms of the NCS groups and two further N atoms of the positively charged DABCO ligands, leading to filamentous molecules  distances of 2.350 (2) Å, the octahedron is considerably elongated. This can be explained through the steric demand of the DABCO+ units.The asymmetric unit consists of a Coon Fig. 1, with thes Fig. 2. The Co—n-dodecyl chains aligned parallel to each other 4]. 1-Dodecyl-4-aza-1-azoniabicyclo[2.2.2]octane chloride (Dodeca-DABCO-Cl) was prepared by the reaction of DABCO  with 1-chlorododecane  in 20 ml of acetonitrile. The mixture was refluxed for 10 h and the solvent removed under reduced pressure. Potassium tetra-(isothiocyanato)cobaltate(II) was prepared through the reaction of potassium isothiocyanate  with cobalt(II) chloride  in 250 ml of acetone. The mixture was refluxed for 2 h, the solvent removed and the raw product extracted with ethyl acetate in a soxhlet extractor. Dodeca-DABCO-Cl  and K2[(Co(NCS)4]  were mixed in 10 ml of acetonitrile and stirred for 1 d at ambient temperature. The mixture was filtered and the solvent removed under reduced pressure. The resulting blue solid was washed several times with acetone. Large blue crystals were grown by leaving the flask open and allowing the solvent acetonitrile to evaporate over the course of three days. The melting point is 331 K.The compound is accessible through the reaction of 1-dodecyl-4-aza-1-azoniabicyclo[2.2.2]octane chloride with KCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620000231/wm4120sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314620000231/wm4120Isup2.hklStructure factors: contains datablock(s) I. DOI: 1976544CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-022-27237-0, published online 29 December 2022Correction to: +TIL (1/2/3)” were swapped with the values in row “Combined index of PD-L1 and stromal CD8+TIL (1/2/3)” in columns “PFS” and “OS”. The correct and incorrect Tables appear below.The original version of this Article contained an error in Table 3, where the values in row “Combined index of PD-L1 and tumoral CD8Incorrect:Correct:The original Article has been corrected.


In the molecule, the four rings are not in the same plane, the pyridine ring being inclined to the benzene rings by 17.26 (6), 56.16 (3) and 24.50 (6)°. In the crystal, molecules are linked by C—H⋯π interactions into a three-dimensional network.The title compound was obtained  24H19NO, was obtained via the reaction of -3-(3-methoxyphenyl)-1-phenylprop-2-en-1-one with ethyl 2-oxopropanoate, using NH4I as a catalyst. The compound crystallizes in the monoclinic space group I2/a. In the molecule, the four rings are not in the same plane, the pyridine ring being inclined to the benzene rings by 17.26 (6), 56.16 (3) and 24.50 (6)°. In the crystal, molecules are linked by C—H⋯π interactions into a three-dimensional network. To further analyse the intermolecular interactions, a Hirshfeld surface analysis was performed. Hirshfeld surface analysis indicates that the most abundant contributions to the crystal packing are from H⋯H (50.4%), C⋯H/H⋯C (37.9%) and O⋯H/H⋯O (5.1%) interactions.The title compound, C Given this background, we report herein the synthesis and crystal structure of the title compound, which was synthesized by NH4I-triggered annulation of α,β-unsaturated ketoxime acetates.Substituted pyridines are privileged scaffolds in medicinal chemistry and are versatile building blocks for the construction of natural products  ring motif.The title compound crystallizes in the monoclinic crystal system in space group 3.Cg2i and C24—H24⋯Cg3ii, Cg2 and Cg3 are the centroids of the C6–C11 and C12–C17 rings, respectively, symmetry codes as in Table 1Cg3 interactions generate stacks along the b-axis direction. These stacks are linked by the C14—H14⋯Cg2 interactions. The packing is strengthened by van der Waals interactions between parallel molecular layers.In the crystal Fig. 2, the molCrystal Explorer  close contacts. Fig. 4a is the fingerprint plot showing all intermolecular interactions while Fig. 4b–d show these resolved into C⋯H/H⋯C (37.9%), H⋯H (50.4%) and O⋯H/H⋯O (5.1%) contributions, respectively. As a result, van der Waals interactions are dominant in the crystal packing.In order to investigate the intermolecular interactions in a visual manner, a Hirshfeld surface analysis was performed using 4.et al., 2016viz. 4-(4-fluorophenyl)-2,6-diphenylpyridine [(I) SURGER01; Zhang et al., 2021et al., 2019et al., 1994et al., 2011et al., 2017et al., 2017et al., 2009et al., 2018A search of the Cambridge Structural Database 5.E,2E)-3-(3-Methoxyphenyl)-1-phenylprop-2-en-1-one (3.0 mmol), ethyl 2-oxopropanoate (0.3 mmol), NH4I  and NaHSO3  were loaded into a 20 mL tube under an N2 atmosphere. The solvent toluene (15 mL) was added into the tube by syringe. The reaction mixture was stirred at 373 K for 12 h. Upon completion of the reaction, the mixture was then allowed to cool down to room temperature and flushed through a short column of silica gel with EtOAc (15 mL). After rotary evaporation, the residue was purified by column chromatography on silica gel (petroleum ether/EtOAc) to give the product as a white solid. Part of the purified product was redissolved in petroleum ether/ethyl acetate and colourless crystals suitable for X-ray diffraction were formed after slow evaporation for several days. Spectroscopic data: 1H NMR  δ 8.20 , 7.87 , 7.53–7.50 , 7.46–7.42 , 7.33–7.32 , 7.26–7.24 , 7.02–7.00 , 3.89 ; 13C NMR  δ 160.2, 157.5, 150.2, 140.6, 139.5, 130.2, 129.1, 128.8, 127.2, 119.7, 117.3, 114.3, 113.1, 55.5.(16.Uiso(H) = 1.2Ueq (C) or 1.5Ueq(CMe) was applied in all cases.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022007812/pk2666sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022007812/pk2666Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022007812/pk2666Isup3.cmlSupporting information file. DOI: 2194417CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E. coli coexpressing methylmalonyl CoA synthase (MatBrp), methylmalonyl CoA reductase (MCR) and malonate transmembrane protein (MadLM). The concentration of 2-F-3-HP reached 50.0 mg/L by whole-cell transformation after 24 h. 2-F-3-HP can be used as the substrate to synthesize other fluorides, such as poly (2-fluoro-3-hydroxypropionic acid) (FP3HP). Being entirely biocatalytic, our procedure provides considerable advantages in terms of environmental and safety impacts over reported chemical methods.Fluorine has become an important element for the design of synthetic molecules for use in medicine, agriculture, and materials. The introduction of fluorine atoms into organic compound molecules can often give these compounds new functions and make them have better performance. Despite the many advantages provided by fluorine for tuning key molecular properties, it is rarely found in natural metabolism. We seek to expand the molecular space available for discovery through the development of new biosynthetic strategies that cross synthetic with natural compounds. Towards this goal, 2-fluoro-3-hydroxypropionic acid (2-F-3-HP) was first synthesized using  Organic fluorides are important compounds that are widely used in the fields of pharmaceuticals, molecular imaging, and materials . FluorinDespite the many advantages provided by fluorine for tuning key molecular properties, it is rarely found in natural metabolism, which limits the application of fluorine-containing compounds . AccordiE. coli  is an important platform compound with a wide range of applications . It is eE. coli coexpressing methylmalonyl CoA synthase (MatBrp) from Rhodopseudomonas palustris, methylmalonyl CoA reductase (MCR) from Thermophilic Filiculture and malonate transmembrane protein (MadLM) from Pseudomonas pf5 with 2-FMA as substrate  as the initial fluorine source. 2-FMA is both relatively inexpensive and capable of being produced through enzymatic pathways while avoiding the acute organofluorine poisoning arising from the direct application of fluoroacetate to cells . After wubstrate . The advubstrate . Theoretubstrate , could aE. coli BL21 (DE3) competent cells were purchased from Vazyme .2-FMA and other chemicals were obtained from Aladdin . A DNA gel extraction kit, plasmid purification kit, Primer STAR Max and DNA marker were obtained from TAKARA (Japan). Protein markers and T4 DNA ligase were obtained from Thermo Fisher Scientific (United States). M9 Minimal Salts (M9 buffer) were obtained from Sangon Biotech . MatBrp (WP_011155789.1), Mcr (AAS20429.1), MadL (AAY95003.2), MadM gene (WP_011063986.1) were synthesized by Beijing Genomics Institute . The synthesized MatBrp was digested with BglII, and the synthesized Mcr was digested with BglII and BamHI. The digested MatBrp and Mcr were ligated into the pET28a vector, which was digested with NcoI, EcoRV and KpnI. The MadLM gene was digested with BglII and XbaI, and the digested fragment was ligated into the pBAD, which was digested with the EcoRI, XbaI and HindIII. The constructed vector was transformed into E. coli BL21 (DE3).The E. coli cells BL21 (DE3) harboring recombinant plasmids were incubated at 37 °C for 12 h in 5 ml of LB medium . The grown cells (5 ml) were then transferred into 50 ml of LB medium  and cultivated at 37 °C in the thermostatic incubator . When OD600 nm reached 0.6, 0.5 mM isopropyl beta-d-1-thiogalactopyranoside (IPTG) and 0.02% arabinose were added. The cells were harvested by centrifugation  at 6,000 rpm for 5 min and washed with 100 mM Tris-HCl . After the cells (10 g of wet weight) were disrupted by a high-pressure cell cracker , the cell debris was discarded, the supernatant (crude enzyme solution) was obtained by centrifugation . The standard assay was as followed: 1 mg/ml crude enzyme, 20 mM 2-FMA, 2 mM NADPH, 2 mM ATP, 1 mM CoA, 100 mM Tris-HCl buffer (10 mM MgCl2), pH 7.8, 30 °C, 200 rpm, 12 h. The reaction solution was detected by LC-MS.E. coli cells BL21 (DE3) harboring recombinant plasmids were incubated at 37 °C for 12 h in 5 ml of LB medium containing appropriate antibiotics. The grown cells (5 ml) were then transferred into 50 ml of LB medium containing appropriate antibiotics and cultivated at 37 °C in the thermostatic incubator. When OD600 nm reached 0.6, 0.5 mM IPTG and 0.02% arabinose were added.2HPO4·12 H2O, 3 g/L KH2PO4, 0.5 g/L NaCl and 1 g/L NH4Cl, pH 7.0) to remove residual culture media and further resuspended in M9 buffer. The typical assay to directly measure reaction products was performed as follows: wet whole cells (OD600 nm = 30), 4 mM 2-FMA, 8% glucose and 10 mM MgSO4. Reactions were performed in M9 buffer (pH 7.0) at 30°C with persistent stirring at 200 rpm. After centrifugation, the supernatants were analyzed by HPLC.The cells were washed with M9 buffer (15.12 g/L Na2O (0.01% formic acid); B: acetonitrile; Column: Acclaim Organic Acid ; column temperature: 25°C; velocity of flow: 0.2 ml/min; linear gradient: 0% B, 0–4 min; 0%–30% B, 4–20 min; 30%-80%B, 20–22 min; 80% B, 22–26 min; 80%–0% B, 26–27 min; 0%, 27–32 min. Injection volume: 1 µL. MS conditions: Source: ESI; Scan type: negative, full scan; Scan range (m/z): 15–300; Fragmentor (V): 50; Cell Accelerator Valtage: 3; Capillary: 4.0 KV; Gas Temp: 350°C; Gas Flow: 9 L/min; Nebulizar: 35 psi; 0–2 min, LC to waste; 2–32 min, LC to MS.LC-MS conditions: ultra-high pressure liquid chromatography-triple quadrupole mass spectrometer ; Mobile phase: A: H2SO4; Column: HPX-87H ; column temperature: 50°C; velocity of flow: 0.5 ml/min; Injection volume: 5 µL.HPLC conditions: High-performance liquid chromatography ; Mobile phase: 5 mM H2O, transfer to a nuclear magnet tube to determine the chemical structure of 2-F-3 HP by fluorine spectrometry (19F-NMR).The fermentation broth is centrifuged for 5 min, the supernatant is collected, and the spin distillation concentrates to 1 ml. Take 500 μL of concentrate, add 10 μL of trifluoroacetic acid as the internal standard, add 100 μL DIn this study, 2-FMA was used as a substrate to biosynthesize 2-F-3-HP by the catalysis of MatBrp and MCR. The synthetic process of the 2-FMA was simple and mild. On the one hand, 2-FMA can be synthesized by microorganisms . On the Pseudomonas can transport 2-FMA into cells and improve the yield of fluoromalonyl-CoA  was collected, and soluble expression levels of MatBrp and MCR were detected by SDS-PAGE. Compared with the control strain 1 (BL21 (DE3)/pACYCDute1/pBAD), MatBrp (55 kDa) and MCR (135 kDa) were expressed in the recombinant strain 2 /pACYCDuet1-strain 2 . The solUsing the crude enzyme of the recombinant strain as the catalyst, and NADPH, ATP and coenzyme A as the donors, the purpose was to verify that 2-FMA could be catalyzed by MatBrp and MCR to generate 2-F-3-HP. The formation of 2-F-3-HP was analyzed by HPLC-MS and the results are shown in 19F-NMR, and all fluorometabolites present in the samples were scanned. The same peak  as that of the standard was detected in the sample, which indicated that 2-F-3-HP was generated in the sample  (FP3HP) or poly (2-fluoro-3-hydroxypropionic acid)-block-poly (2-fluoro-3-hydroxybutyric acid) (FP3HB-b-FP3HP), which is expected to obtain fluorine-containing materials with better properties.In this study, 2-F-3-HP was first synthesized by the engineered


The data suggest a promising role for anti-IL-6 receptor antibody and anti-TLR2 receptor antibody treatment for oral cancer.In an endeavour to understand metastasis from oral squamous cell carcinomas, we characterised the metastatic potential of a human tongue derived cell line (SCC-4 cells) and compared this phenotype to pre-cancerous dysplastic oral keratinocyte (DOK) cells derived from human tongue and primary gingival keratinocytes (PGK). We demonstrate that SCC-4 cells constitutively synthesize and release significant amounts of IL-6, a process that is enhanced by the addition of the TLR2/TLR6 agonist, Pam2CSK4. The expression of TLR2/6 and IL-6Ra/gp130 receptors was also confirmed in SCC-4 cells. Cancerous SCC-4 human tongue cells also have a classic EMT profile, unlike precancerous human tongue DOK cells. We also established that IL-6 is driving The online version contains supplementary material available at 10.1007/s12032-022-01664-5. Squamous cell carcinoma (SCC-4) cells were originally established from the tongue of a 55-year-old male [SCC-4 cat# ECACC 89062002]. Mycoplasma-free cells were grown in cell culture flasks in Dulbecco’s Modified Eagle’s Medium GlutaMAX cell culture medium (Gibco) supplemented with 5 µg/ml hydrocortisone, 20% (v/v) Foetal Bovine Serum (FBS) and penicillin–streptomycin (50 U/ml and 50 μg/ml) (Gibco). Cells were grown at 37 °C in humidified environment containing 95% O® (Invitrogen) was employed to quantitatively measure the viability of cells. Resazurin, the active cell-permeable ingredient of alamarBlue®, has a blue colour and after entering the cells, is reduced to resorufin, which then produces a red fluorescence. Viable cells are able to convert resazurin to resorufin giving a quantitative measure of their viability, while non-viable cells with a worse innate metabolic activity produce less resorufin. Plates were read on a Spectramax Gemini Plate Reader using SOFTmax Pro version 4.9  at excitation and emission wavelengths of 544 nm and 590 nm, respectively. Experiments were conducted in triplicate and RFU values were expressed as mean ± SEM of the three experiments.AlamarBlue2SO4) was added and OD values were obtained by measure absorbance at 450 nm using Spectramax Microplate Reader (Molecular Devices). Concentrations of cytokines were determined using the standard curve from each ELISA plate. Commercial Human ELISA kit information: IL-6, (Biolegend/MSC); IL-8 (Biolegend/MSC); TNF-α (Biolegend/MSC); IL-11 (R&D Systems).Commercially available ELISA kits were used according to manufacturer’s instructions. Once sufficient colour was developed, 20 μl/well stop solution (2 N H. [Quantification of protein concentrations in cell lysates was carried out using the Bicinchoninic Acid (BCA) Assay as described by Smith et al. .. [1, Clone 17506 (R&D Systems); gp130 (MAB2281), Mouse monoclonal IgG1, Clone 29104 (R&D Systems); Vimentin (5741), Rabbit monoclonal, Clone D21H3 XP® ; E-cadherin (3195), Rabbit monoclonal, Clone 24E10® ; N-cadherin (13116), Rabbit monoclonal, Clone D4R1H XP® ; β-catenin (8480), Rabbit monoclonal, Clone D10A8 XP® ; β-actin (4970), Rabbit monoclonal ; GAPDH (CB1001) Rabbit monoclonal ; HRP conjugated anti-goat IgG (705-035-003) (Jackson Immunolabs); HRP conjugated anti-rabbit IgG (W401)(Promega).SDS-PAGE and immunoblotting were performed as previously described by Geoghegan et al. . AntibodDensitometric analyses of protein expression on western blots were performed using the Image Lab Software, Bio-Rad Laboratories.5 cells/ml per well and allowed to attach overnight. Upon determination of the optimal antibody concentrations, DOK and SCC-4 cells were treated with 30 ng/ml rhIL-6, 10 μg/ml IL-6 neutralising mAb, 40 μg/ml IL-6Ra mAb, combination of 30 ng/ml rhIL-6 and 40 μg/ml IL-6Ra mAb, 200 ng/ml Pam2CSK4, combination of 200 ng/ml and 10 μg/ml anti-TLR2 neutralising antibody. Complete medium and IgG2B isotype control for the IL-6 neutralising mAb were used as control. 24 h later, 50 µl of alamarBlue® dye  was added to each well and incubated again at 37 °C/5% CO2 for 3.5 h. Fluorescence was measured at excitation and emission wavelengths of 544 nm and 590 nm, respectively, using the Spectramax Gemini Plate Reader using SOFTmax Pro version 4.9 . Antibody information: Human anti-IL-6Ra (MAB227), Mouse monoclonal IgG1, clone 17506 (R&D systems); Human IL-6 (MAB2061), 1936 (R&D systems); Isotype Control (MAB004), mouse monoclonal IgG2B, clone 20116 (R&D systems); Human TLR2 antibody (HM1054), mouse monoclonal IgG1, Clone T2.5 (Hycult Biotech).The assay was performed was essentially as described by McNamee and O’Driscoll . Tissue t-test, while for comparisons of more than two groups, data were analyzed using one-way or two-way ANOVA followed by Tukey’s or Sidak’s or Bonferroni’s multiple comparison tests were performed. For all comparisons, p-value of *p < 0.05, **p < 0.01, ***p < 0.001 were considered to be significant.Statistical analyses were performed using the computer based mathematical package Graph Pad Prism 8.0 software. All results were expressed as mean ± standard error of the mean (SEM). For comparisons of two groups data were analyzed using a two-tailed unpaired student’s n)).Having optimized conditions for TLR ligand-dependent cytokine secretion , IL-6, IL-11, TNF-α and IL-8 secretion was determined, 24-h after TLR ligand stimulation of primary Gingival Keratinocytes (PGK), pre-cancerous Dysplastic Oral Keratinocytes (DOK) and cancerous Oral Squamous Cell Carcinoma 4 Cells (SCC-4). Figure p = 0.52), when compared with the untreated DOK cells (325.3 ± 6.4 pg/ml (3)), while a significant decrease was observed after Pam3CSK4 (286.9 ± 7.6 pg/ml (3), p = 0.03) and LPS (287.3 ± 0.2 pg/ml (3), p = 0.03) stimulation. On the contrary, IL-11 secreted from untreated SCC-4 cells (92.2 ± 7.5 pg/ml (3)) was significantly lower than that from untreated PGK (274.6 ± 64.2 pg/ml (3)) and DOK cells (325.3 ± 6.4 pg/ml (3)). Interestingly, IL-11 was shown to be significantly increased after stimulation with Pam2CSK4, Pam3CSK4 and LPS ligand in SCC-4 cells (152.7 ± 0.7 pg/ml (3), p < 0.0001, 118.3 ± 5.1 pg/ml (3), p = 0.02 and 118.9 ± 2.1 pg/ml (3), p = 0.02, respectively), while Pam3CSK4 stimulation decreased IL-11 secretion almost fivefold from PGK cells compared to the untreated control group. Pam2CSK4 stimulation did not show any effect on IL-11 release in PGK cells (205.8 ± 26 pg/ml (3)), while no release of IL-11 was observed from the same cell line after LPS stimulation suggesting that this cytokine is not present in the medium (mean ± SEM (n)).Regarding the secretion of another member of the IL-6 family, namely IL-11  and SCC-4 cells (412.9 ± 54.4 pg/ml (3), p = 0.038) exhibited a significant increased secretion compared to their untreated groups, while Pam2CSK4 treated PGK cells (591.2 ± 154.5 pg/ml (3), p = 0.2) slightly but not significantly increased IL-8 production. Furthermore, after Pam3CSK4 stimulation, no significant difference was detected between the treated (374.1 ± 14.6 pg/ml (3), p = 0.99) and untreated control group (347.6 ± 18.7 pg/ml (3)) in PGK cells, while IL-8 production was almost undetectable in both DOK (0.85 ± 0.5 pg/ml (3)) and SCC-4 cells (45.2 ± 16.8 pg/ml (3)). All cells were also treated with LPS, although no significant difference was found between the untreated and treated groups in both DOK (196.2 ± 37.8 pg/ml (3), p = 0.7) and SCC-4 (297.2 ± 123.6 pg/ml (3), p = 0.2), while IL-8 was not detected in LPS-treated PGK cells, suggesting that this cytokine is not produced (mean ± SEM (n)). Figure Untreated PGK cells secreted the highest IL-8 levels (347.6 ± 18.7 pg/ml (3)) compared to untreated DOK (118.2 ± 30.8 pg/ml (3)) and untreated SCC-4 cells (85.5 ± 12.2 pg/ml (3)) ).Having established the optimal concentration of anti-TLR2 blocking antibody in DOK and SCC-4 cells , the effect of anti-TLR2 antibody (10 μg/ml) on IL-6 release was analysed in these cell lines  [anoikis resistance is a form of apoptosis in DOK and SCC-4 cells, adherent and in suspension (poly-HEMA), cells were examined by flow cytometry analysis of annexin V/PI-stained cells. Cells grown under anchorage—independent conditions (poly-HEMA) were observed to have induced apoptotic cells death compared to necrotic cells. DOK cells (poly-HEMA) were found to have the highest apoptotic rate . In this way, using the anoikis resistance assay, or anchorage-independent growth assay, survival rate of detached DOK and SCC-4 cells were examined from their artificial substratum. The anoikis resistance for the untreated groups was significantly lower in DOK  and 63.8% ± 3.3 (5), respectively). Interestingly, the anoikis survival rate was significantly enhanced in SCC-4 cells after rhIL-6 addition and Pam2CSK4 addition (88% ± 4.9 (6) and 82.9% ± 4.8 (3), respectively), while no significant effect of rhIL-6 addition (40.9% ± 3.7 (9)) or Pam2CSK4 addition (42.1% ± 1.8 (5) was observed, compared to untreated and isotype controls, in DOK cells. However, anoikis survival rate was significantly diminished upon treatment of SCC-4 cells with monoclonal IL-6 neutralising and monoclonal IL-6Ra antibodies (35.6% ± 4.7 (3) and 25.8% ± 5.5 (3), respectively), while no significant differences were observed in DOK cells treated with IL-6 neutralising and IL-6Ra antibodies (24% ± 3.3 (3) and 29.1% ± 2.2 (3), respectively). No major effect was observed upon treatment with monoclonal IL-6Ra in combination with exogenous rhIL-6 in both DOK and SCC-4 cells (27.2% ± 5.1 (3) and 56.9% ± 3.2 (3), respectively), while combined treatment with Pam2CSK4 and anti-TLR2 neutralising antibody significantly decreased the survival rate in SCC-4 cells (34.3% ± 4.1 (5)), while no major change was shown again in DOK cells (29.5% ± 2.9 (4)).Metastatic cancer cells are able to survive and resist optosis) . One can. [Of the cytokines investigated, IL-6 was the most abundant cytokine produced by our cancerous cells which led us to give it some focus in this study. It was demonstrated that IL-6 secretion is threefold higher from SCC-4 cells compared to normal PGK, while IL-6 secretion is undetectable from DOK cells. This observation is consistent with that of Chen et al.  who obse.  also sug. , 28.. [Our investigation progressed to determine whether TLR ligand activation had an effect on IL-6 production levels from the cells used in this study. When SCC-4 cells were treated with the TLR2/TLR6 agonist, Pam2CSK4, there was a significant threefold increase in IL-6 production in both SCC-4 and PGK cells. No significant increase in IL-6 production was observed with TLR1/TLR6 ligand, Pam3CSK4. Interestingly, Chuang et al.  examined.  which maBy contrast, it was revealed that both PGK and DOK cells IL-11 secretion levels were threefold higher when compared to SCC-4 cells. Interestingly, only Pam3CSK4 and LPS agonists slightly but significantly affected IL-11 secretion levels in DOK cells, while only Pam3CSK4 significantly diminished IL-11 production in PGK cells. Significant IL-11 increase was also found in SCC-4 cells after stimulation with all agonists but in general IL-11 levels were significantly lower than the levels in treated dysplastic counterparts. Elevated levels of IL-11 (~ 250 μg/l) have been identified in breast cancer patients which is associated with bone metastasis .TNF-α, a widely expressed pleiotropic cytokine, is increased during inflammation and cancer in saliva and patient sera. Indeed, it has been reported that TNF-α levels might act as diagnostic markers for detection of oral cancer since it is more abundant in saliva than in plasma , 33 and IL-8 has been found in the tumour microenvironment in a range of different cancer types, including OSCC tumours. Clinical studies have reported elevated salivary IL-8 levels in OSCC patients , 36 compTLRs have been reported to be expressed in various types of cancers with key roles in carcinogenesis and tumour progression . Since PIn support of these findings, a previous report revealed higher TLR2 expression in the microenvironment of the keratinocytes of dysplastic epithelium and OSCC, when compared to hyperplasic cells . InteresAfter observing the effect of TLR2/6 ligand on IL-6 secretion from DOK and SCC-4 cells and the levels of TLR2 expression in these cells, it was decided to investigate whether there was expression of receptors for IL-6, or more particularly expression of IL-6Ra and gp130, in DOK and SCC-4 cells using western blotting. These findings suggest significantly higher IL-6Ra and gp130 expression in SCC-4 cells compared to DOK cells, regardless of the stimulation with rhIL-6. This result suggests that classical IL-6 signaling occurs in SCC-4 cells since both receptors were expressed, and consequently IL-6 is able to bind to the membrane-bound receptor IL-6Rα, which has the capacity for heterodimerization with gp130 and thus forming a complex that allows the JAK/STAT signaling cascade to operate .Anoikis resistance, markers of EMT, and cell death were characterized in these cells. Evidence is provided for IL-6 playing a central role in anoikis resistance. Data from this study show that DOK cells displayed non-EMT characteristics, expressing E-cadherin and lower levels of β-catenin compared to SCC-4 cells. High levels of vimentin and N-cadherin were observed in SCC-4 cells compared to DOK cells, while complete loss of E-cadherin was demonstrated in SCC-4 cells, confirming the EMT phenotype of these cells. Moreover, it was demonstrated by Wu et al. [The effect of endogenous and exogenous IL-6 on cancer phenotypes of pre-cancerous human tongue DOK cells and cancerous SCC-4 human tongue cells were also investigated. u et al.  that IL-u et al. . Similaru et al. . Anotheru et al. , lymph nu et al. , while aassociated with the regulation of tumour proliferation and metastasis . HoweverAnoikis resistance, or the ability for cells to live detached from the extracellular matrix, is a property of epithelial cancers. In this study, resistance to anoikis was assessed in DOK and SCC-4 cells. The results demonstrated that SCC-4 cells showed significantly greater resistance to anoikis when compared to DOK cells. In particular, half the population of untreated dysplastic DOK cells (49.2%) were able to resist anoikis when detached from the ECM, while survival rates of untreated cancerous SCC-4 cells were significantly higher (1.5-fold at 76.5%) as might be expected from cancerous cells. It was then decided to look at the effect of IL-6 on anoikis resistance in DOK and SCC-4 cells. The results indicated that rhIL-6 treatment significantly enhanced (1.4-fold) resistance to anoikis in SCC-4 cells when compared to untreated control levels, an observation consistent with that seen by Fofaria and Srivastava [ivastava  for pancanoikis resistance in SCC-4 cells was increased (1.3-fold) upon stimulation with Pam2CSK4, an observation that is consistent with a role for endogenous IL-6 being important for anoikis, and consistent with the observation that Pam2CSK4 triples IL-6 secretion from SCC-4 cells. Again, consistent with a role for endogenously produced IL-6 in anoikis, is the observation that treatment of SCC-4 cells with IL-6 receptor (IL-6Ra) monoclonal antibody significantly reduced (2.5-fold) anoikis resistance, a result that is further endorsed by the observation that neutralising IL-6 or blocking TLR2 with the monoclonal antibodies also reduces anoikis resistance in SCC-4 cells (1.8-fold). Consistent with these observations is the observation that anoikis resistance was not affected when extrinsic IL-6 was added in combination with IL-6Ra addition. There was no effect of rhIL-6 addition, IL-6Ra addition, IL-6 neutralizing antibody, TLR2 blocking antibody or a combination of rhIL-6 plus IL-6RA on anoikis resistance in pre-cancerous dysplastic DOK cells.Similarly, anoikis resistance. The quantitatively large increase in IL-6 production from SCC-4 cells with addition of the Pam2CSK4, but not Pam3CSK4, is consistent with our data confirming the existence of TLR2/6 and IL-6Ra/gp130 receptors in SCC-4 cells. The fact that DOK cells do not produce IL-6 is consistent with the lack of IL-6Ra/gp130 receptors in DOK cells. The role of TLR2 on enhanced IL-6 production is further cemented by the observation that anti-TLR2 receptor blocking antibodies reduced, by half, the IL-6 production in SCC-4 cells and significantly reduces anoikis resistance. The central role played by IL-6 in anoikis in SCC-4 cells is also demonstrated by the observations that added recombinant IL-6 increases anoikis resistance, and that IL-6 neutralizing antibody and IL-6 receptor antibodies reduce anoikis resistance. This study provides evidence, for the first time, that IL-6 drives anoikis resistance in SCC-4 cells.We show that a quantitatively significant amount of IL-6 is secreted by oral cancerous SCC-4 cells. In addition, the TLR2 ligand Pam2CSK4 enhances IL-6 secretion from SCC-4 cells and enhances Supplementary file1 (PDF 785 KB)Below is the link to the electronic supplementary material.


ATN-mG/P exhibited greatly boosted apoptotic activity in 4T1 cells, induced potent immunogenic cell death (ICD), and efficiently stimulated maturation of bone marrow-derived dendritic cells (BMDCs). Remarkably, in a postoperative TNBC model, ATN-mG/P combining with NanoCpG promoted strong anti-cancer immune responses, showing a greatly augmented proportion of mature DCs and CD8+ T cells while reduced immune-suppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Treg), which led to complete inhibition of lung metastasis and 60% mice tumor-free. The co-delivery of gemcitabine and paclitaxel at desired ratio in combination with NanoCpG provides a unique platform for potent chemoimmunotherapy of “cold” tumors like TNBC.Triple-negative breast cancer (TNBC) due to lack of clear target and notorious “cold” tumor microenvironment (TME) is one of the most intractable and lethal malignancies. Tuning “cold” TME into “hot” becomes an emerging therapeutic strategy to TNBC. Herewith, we report that integrin-targeting micellar gemcitabine and paclitaxel .•ATN-mG/P facilitates integrin-targeted and synergistic tumor cell ICD and orchestrated APC regulation in TME.•ATN-mG/P combined with NanoCpG further heats up immune microenvironment.•The combo brings survival benefits to postoperative TNBC mice with 60% cure.•The combo provides a strategy to potent chemoimmunotherapy of “cold” tumors. A coupl therapy . For exa therapy . Zhang e therapy . The com therapy , though  therapy .+ T cells, and repolarize tumor-associated macrophages (TAMs) towards M1 phenotype, leading to significantly inhibited TNBC primary tumor and metastases [The “cold” tumors might also be “heated up” by immunoadjuvants such as toll-like receptors (TLR) agonist , indoleamine 2,3-dioxygenase (IDO) inhibitor  and stimulator of interferon genes (STING) agonist cyclic dinucleotide . Among ttastases . Ran et tastases . We recetastases ,25. It sHerewith, we report that integrin-targeting micellar gemcitabine and PTX (ATN-mG/P) cooperating with NanoCpG effectively “heated up” and treated postoperative TNBC mouse model . ATN-mG/22.1HPG and PTX were dissolved separately in PEG350 (25 mg/mL) and blended at a molar ratio of 20/1, 10/1 or 5/1. Drug solution  was then mixed with 5 μL PEG350 solution of PEG-P(CL-DTC) (200 mg/mL), and PEG350 was added to a final volume of 50 μL. Such mixture was then injected under stirring into 950 μL phosphate buffer , yielding mG/P with different molar ratios of HPG and PTX. Similarly, for ATN-mG/P with ATN content of 5%, 50 μL PEG350 solution containing 4.75 μL PEG-P(CL-DTC) (200 mg/mL), 2 μL ATN-PEG-P(CL-DTC) (25 mg/mL), HPG/PTX solution and PEG350 were added to 950 μL PB. The size, size distribution and stability of mG/P and ATN-mG/P were determined by dynamic light scattering (DLS). The drug loading and release of mG/P and ATN-mG/P as well as the intact HPG in free HPG, mGem and mG/P (172 μM GEM equiv.) in the presence of cytidine deaminase (CDA) and 10% serum were determined by HPLC (detailed in Supporting Information).2.23/well) were added with 20 μL mG/P  with HPG concentration of 0.0017–68.8 μM, or mPTX with PTX concentration of 0.005–12.8 μM. After 48 h incubation, 3--2,5-diphenyltetrazolium bromide (MTT) solution  was added to incubate for 4 h before washing and addition of 150 μL dimethyl sulfoxide (DMSO) to dissolve the purple formazan produced by living cells. The determination of the cell viability and half-maximal inhibitory concentration (IC50) was the same as reported (n = 6) [4T1-luc cells seeded in 96-well plates (1 × 10 (n = 6) .50 of each drug in mG/P, and A and B represent respective the IC50 of each drug in single formulation mGem or mPTX. CI < 1: synergistic effect; CI = 1: additive effect; CI > 1: antagonistic effect.Combination Index (CI) between two drugs was calculated based on the formula3/well), and the cells were then incubated 44 h with drug-free fresh media. The sample processing and data analysis methods are as described above.To investigate the targetability of ATN-mG/P toward 4T1 cells, ATN-mG/P and mG/P at Gem/PTX = 10/1 (HPG conc: 0.0017–68.8 μM) were incubated 4 h with 4T1 cells (1 × 102.35/well) were incubated with ATN-mG/P (Gem/PTX: 10/1), mG/P , mGem, or mPTX , taking PBS as control. After 24 h, the culture medium was collected for determining ATP using enhanced ATP assay kit, and the cells for detection of CRT. To determine CRT exposure, these cells were digested, added with αCRT for 1 h, and incubated with Alexa 647-conjugated secondary antibody for 30 min. Between two steps, washing  was applied. The cells were then measured using flow cytometry and data were analyzed using FlowJo_V10 to determine the CRT expression.4T1-luc cells cultured 24 h in 12-well plates (1 × 102.46/well) were incubated with ATN-mG/P (Gem/PTX: 10/1), mG/P , mGem, or mPTX , taking PBS as control. After 24 h, the cells were centrifuged, washed and incubated with FITC-αCD11c, APC-αCD80 and PE-αCD86 for 30 min. Then the cells were immediately measured by flow cytometry and analyzed using FlowJo_V10 to quantify mature BMDCs (CD11c+CD80+CD86+ mDCs).BMDCs cultured in 12-well plates (1 × 106/well) and 4T1-luc cells (1 × 105/well) were separately cultured in 12-well plates for 24 h. The culture medium of 4T1-luc cells were removed, and BMDCs were added. The two types of cells were then co-incubated with ATN-mG/P (Gem/PTX: 10/1), mG/P , mGem, or mPTX  for 24 h. The following treatment and measurement were as described above (n = 3).To study the effect of NanoCpG on BMDC maturation, NanoCpG, free G/P (mixture of free HPG and PTX at molar ratio of 10/1), mG/P, ATN-mG/P, mG/P + NanoCpG, and ATN-mG/P + NanoCpG  were added to BMDCs. The following treatment and measurement were as described above (n = 3). To study the maturation of BMDCs co-culture with 4T1 cells, BMDCs (1 × 102.55/mouse with 30 vol.% Matrigel) subcutaneously in the right upper hind leg of Balb/c mice . After 7 days the 4T1-luc tumor volume reached ca. 50 mm3 (designated as day 0), and the mice were randomly divided into five groups (n = 6): PBS, mPTX , mGem, mG/P (Gem/PTX: 20/1 or 10/1) at HPG dose of 15 mg/kg (25.8 μmol/kg). On day 0, 2, 4, 6, 8, and 10, drug formulations were intravenously administered into the mice. The tumor volume and body weight were monitored every two days. The survival rates of the mice were recorded and the mice were also considered dead at tumor volume over 2000 mm3.All animal experiments were approved by the Animal Care and Use Committee of Soochow University (P.R. China), and all protocols conformed to the Guide for the Care and Use of Laboratory Animals. 4T1-luc subcutaneous mouse model was built by injecting 50 μL of 4T1-luc cells (3 × 10+CD80+CD86+), and with FITC-αCD11b and PE/Cy7-αGr-1 to determine the content of MDSCs (CD11b+Gr-1+), followed by the flow cytometric analyses.To study the therapeutic efficacy of ATN-mG/P and the immune microenvironment of tumors, ATN-mPTX, ATN-mGem, mG/P, ATN-mG/P , PTX: 1.5 mg/kg (1.72 μmol/kg)) were i.v. injected into 4T1-luc subcutaneous model, using the same schedule. Four days after last injection (D 14), three mice from each group were euthanized to analyze the infiltration of mDCs and MDSCs in tumor. Tumor tissue was extracted from tumor-bearing mice, ground, centrifuged to obtain single-cell suspension and red blood cells were lysized by red blood cell lysate (ACK). The cells were incubated 30 min at 4 °C with FITC-αCD11c, APC-αCD80 and PE-αCD86 to determine the content of mDCs (CD11c2.63) of subcutaneous 4T1 model as mentioned above. The tumors relapsed quickly and lung metastasis was observed. At seven days after surgery, the relapsed tumor grew to ca.100 mm3 (day 0), and the mice were assigned into six groups (n = 6): PBS, free G/P, mG/P, ATN-mG/P, mG/P + NanoCpG, or ATN-mG/P + NanoCpG , PTX: 2.25 mg/kg (2.58 μmol/kg), CpG: 1.0 mg/kg). On day 0, 2, 4, 6, 8, 10, free G/P, mG/P and ATN-mG/P was injected intravenously. For two combination groups, NanoCpG was injected on day 1, 3, 5. The tumor volume and body weight were monitored every three days. One mouse in each group was euthanized at five days post the last drug-administration to investigate lung metastasis by bioluminescence imaging and H&E staining of lung slices. Other five mice were for observing survival rates. The mice were deemed dead at tumor volume over 2000 mm3 or body weight loss over 15%.The postoperative recurrent/metastatic 4T1-luc model was established by surgically removing tumor bulk (at volume of 200–300 mm2.73 (day 0), the mice were assigned into five groups (n = 4): PBS, mG/P, ATN-mG/P, mG/P + NanoCpG, or ATN-mG/P + NanoCpG , PTX: 2.25 mg/kg (2.58 μmol/kg), CpG: 1.0 mg/kg). On day 0, 2, 4 mG/P and ATN-mG/P were i.v. injected. For two combination groups, NanoCpG was injected on day 1, 3, 5. Two days after last injection, the mouse plasma was collected to quantify the concentration of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin-10 (IL-10), and the mice were euthanized to study metastasis and immune regulation. The lung was weighed and sliced to count the metastatic nodules. The relapsed tumor was weighed to calculate tumor inhibition rate (TIR). Spleen was weighted and sliced. The lymph node, spleen and tumor were ground, centrifuged to obtain single cell suspensions, and erythrocytes were lysized by ACK. Then the cells were incubated 30 min with corresponding antibodies, i.e. PerpCy5.5-αCD45, FITC-αCD11c, APC-αCD80, PE-αCD86, APC-αCD3, FITC-αCD8, PE-αCD4, Alexa 647-αFoxp3, FITC-αCD11b, Alexa 647-αCD206, PE-αF4-80, PE/Cy7-αGr-1, for determining the contents of mDCs (CD11c+CD80+CD86+), MDSCs (CD11b+Gr-1+), CD4+ T (CD3+CD4+), CD8+ T (CD3+CD8+), and Treg (CD3+CD4+FoxP3+). The flow cytometry measurements were followed to analyze the infiltration of immune cells in tumor and spleen.In the postoperative 4T1-luc model, at relapsed tumor volume of ca.100 mm2.8Data were presented as mean ± standard deviation. The significant differences among groups were determined using GraphPad Prism 9 by one-way ANOVA (Tukey multiple comparison tests). Survival rate was analyzed by Kaplan-Meier technique with a log-rank (Mantel-Cox) test. *p < 0.05 means significant difference, **p < 0.01, ***p < 0.001 and ****p < 0.0001 mean highly significant difference.33.12) reportedly possessed a high affinity to α5β1 integrin overexpressed on tumor cells including breast tumor cells and melanoma cells [vβ3 and αvβ5 integrins [The aim of this study is to tune “cold” TME in TNBC to “hot” therefore enabling efficient immunotherapy, for which we designed integrin-targeting micellar Gem and PTX (ATN-mG/P) to specifically co-deliver both drugs at predetermined ratio to TNBC cells. PTX and Gem both can stimulate ICD. The targeted delivery of Gem and PTX to cancer cells is important to lessen their immune-toxicity. ATN peptide  kg/mol) containing both hydrophobic phosphorylated gemcitabine (HPG) and PTX  in an aqueous solution. Similarly, single drug micelles (ATN-mGem and ATN-mPTX) and non-targeted dual drug micelles (mG/P) were prepared as controls. Notably, all micellar formulations had small sizes (19.8–23.2 nm) and particle dispersity index (PDI) (0.08–0.17) (Here ATN-mG/P was conveniently prepared via co-self-assembly of a PEG350 solution of 5% ATN-PEG-P(CL-DTC) and 95% PEG-P(CL-DTC) (08–0.17) . Interes08–0.17) . The enh08–0.17) , PTX pre08–0.17)  and in P08–0.17) . The sta08–0.17) b. In con08–0.17) c. Notabl08–0.17) . These r3.250 (μM) of 2.3/0.10, 0.4/0.07 and 1.8/0.16 (corresponding to IC50 of HPG and PTX), respectively, compared to those of mGem and mPTX (3.8 and 1.4 μM)  d and e.  1.4 μM) , pointinin vitro and in vivo [50 of ATN-mPTX than non-targeting mG/P at the same Gem/PTX of 10/1  (****p). It is reported that low-dose PTX can promote the maturation and proliferation of DC cells [We investigated the stimulation of dendritic cells (DCs), the most important APCs, by ATN-mG/P. Flow cytometry results demonstrated that mPTX and mGem at low concentrations could both greatly stimulate BMDCs to mature  and intravenously injected with mG/P (Gem/PTX of 20/1 or 10/1), mGem , or mPTX  every two days (mpk: mg/kg)  a. The re: mg/kg) b and c. : mg/kg) b and c. : mg/kg) d. Fig. 3in vivo active-targeting effect and the regulation of TME of 4T1-luc mice, ATN-mG/P and mG/P were intravenously injected using the same schedule. Targeted micelles containing only one drug, ATN-mPTX and ATN-mGem, were used as controls  of the mice a. On 48 + DCs and CD11c+CD80+CD86+ mDCs in recurrent tumors as compared to all other groups (+ cytotoxic T cells (CTL) can attack and kill tumor cells directly. Flow cytometry analysis results confirmed that CD8+ T and CD4+ T cells in spleen and tumor of ATN-mG/P + NanoCpG group were all higher than those in monotherapy groups (reg) in CD4+ T cells that weaken T cell activity in TME was drastically decreased by all treatments  cells, with no reduction in typical immune cells [The great therapeutic effecacy of ATN-mG/P + NanoCpG treatment was manifested by significantly boosted infiltration of total CD11cr groups f and g. eatments i. The MDeatments m, n. Remne cells .Interestingly, ELISA assay results showed that plasma concentrations of IFN-γ and TNF-α were signifacantly stimulated by mG/P + NanoCpG and ATN-mG/P + NanoCpG, compared with mG/P and ATN-mG/P  o, p. IFN4+ and CD4+ T cells, inclined production of TNF-α and IFN-γ, as well as decrease of immune-suppressive MDSCs, Treg and IL-10. This co-delivery of gemcitabine and paclitaxel in combination with NanoCpG adjuvant seems to be a particularly powerful strategy to improvement of the chemoimmunotherapy of “cold” tumors like TNBC.We have demonstrated that integrin-targeting micellar gemcitabine and paclitaxel (ATN-mG/P) cooperating with polymersomal CpG (NanoCpG) can effectively heat up “cold” tumor microenvironment, resulting in potent chemoimmunotherapy of postoperative recurrent/metastatic TNBC model. Remarkably, 3/5 4T1-bearing mice have been cured by ATN-mG/P + NanoCpG group. This exceptional chemo-immunotherapeutic efficacy is likely a result of a collective effect of effective recruitment and activation of DCs, good antigen-presenting to CD8All animal experiments were approved by the Animal Care and Use Committee of Soochow University (P.R. China), and all protocols conformed to the Guide for the Care and Use of Laboratory Animals.Beibei Guo: Formal analysis, Data curation, Writing – original draft. Yan Qu: Formal analysis, Data curation. Yinping Sun: Formal analysis, Data curation. Songsong Zhao: Formal analysis, Data curation. Zhiyuan Zhong: Conceptualization, Supervision. Fenghua Meng: Conceptualization, Supervision, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


N-(4-methoxyphenyl)piperazine crystallize with six independent molecular species in the asymmetric unit. In each compound, multiple hydrogen bonds link these components into complex sheets.Two salts derived from  N-(4-methoxyphenyl)piperazine with 4-methylbenzoic acid and with benzene-1,2-dicarboxylic acid yields the salts 4-(4-methoxyphenyl)piperazin-1-ium 4-methylbenzoate monohydrate, C11H17N2O+·C8H7O2−·H2O (I), and bis[4-(4-methoxyphenyl)piperazin-1-ium] benzene-1,2-dicarboxylate, 2C11H17N2O+·C8H4O42− (II). These salts both crystallize with Z′ = 2, in space groups PPna21, respectively. In compound (I), a combination of four O—H⋯O, four N—H⋯O, one C—H⋯O and one C—H⋯π(arene) hydrogen bonds link the six independent components into complex sheets, within which the two piperazine rings, the two anions, and the two water molecules are related by an approximate, non-crystallographic translation along the b-axis direction. In compound (II), sheets containing R44(18) and R1012(38) rings are formed by the combined action of eight independent N—H⋯O hydrogen bonds. Comparisons are made with the structures of some related compounds.Co-crystallization of  Apart from this difference, the other pairs of corresponding units  are related by an approximate, non-crystallographic translation . Although there are six independent components in the structure, it is possible to select a compact asymmetric unit in which the components are linked by three O—H⋯O hydrogen bonds and two N—H⋯O hydrogen bonds piperazine with benzene-1,2-dicarboxylic acid , is a 2:1 salt that crystallizes in solvent-free form with Z′ = 2 in space group Pna21. A search for possible additional symmetry revealed none. As for compound (I)Compound (II)ca 10°. This behaviour is characteristic of planar and near-planar alkoxyarenes 3.et al., 1998abet al., 2000et al., 1990et al., 1995y, 0.5)  and . The second sub-structure  and . Propagation of these motifs by inversion thus links adjacent [010] chains into a complex sheet lying parallel to (001).The supramolecular assembly of compound (I)5) Fig. 5. The rib5) Fig. 5, inversi5) Fig. 5 to form re Fig. 7 contains1 screw axis, pass through each unit cell, in the domains 0.25 < z < 0.75 and 0.75 < z < 1.25, but there are no direction-specific interactions between adjacent sheets: the C—H⋯O and C—H⋯π(arene) hydrogen bonds all lie within a single sheet.There are eight independent N—H⋯O hydrogen bonds in the structure of compound (II)1) Fig. 8. Two she4.N-(4-methoxyphenyl)piperazine and carboxylic acids piperazine are those of the maleate piperazine, we have recently reported  in methanol (10 ml) was mixed with a solution of the appropriate acid, 4-methylbenzoic acid  for (I)v/v).For the preparation of compounds (I)6.3 or 0.99 Å (CH2), and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms. For the H atoms bonded to N or O atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N) or 1.5Ueq(O), giving the N—H and O—H distances shown in Tables 1Kα radiation was used to facilitate establishing a unique orientation for the structure with respect to the polar axis direction. For the crystal selected for data collection, however, the value of the Flack x parameter  was inconclusive due to its high standard uncertainty, while that calculated using 4511 quotients  − (I−)]/[(I+) + (I−)] was x = 0.45 (5), strongly suggesting the likelihood of twinning by inversion.Crystals of (I)10.1107/S2056989022008337/zl5034sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989022008337/zl5034Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989022008337/zl5034IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989022008337/zl5034Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989022008337/zl5034IIsup5.cmlSupporting information file. DOI: 2202591, 2202590CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Urinary metabolites of vitamin E, i.e., α- and γ-carboxyethyl hydroxychroman (α- and γ-CEHC), have gained increasing attention and have been proposed as novel biomarkers of vitamin E intake and status. However, there are insufficient data on the relationship of plasma α-tocopherol and γ-tocopherol and dietary vitamin E intake with 24 h urinary excretions of α- and γ-CEHC.We aimed to (1) investigate the associations of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC with plasma α- and γ-tocopherol, respectively; (2) investigate the associations of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC with dietary vitamin E intake, and we hypothesize that 24 h urinary excretions of α- and γ-CEHC will better correlate with vitamin E intake than urinary α- and γ-CEHC/creatinine ratios.24 h Urine and plasma samples were collected from 1519 participants  included in the Lifelines-MINUTHE Study for the assessments of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC, and plasma α- and γ-tocopherol. Among those participants, dietary vitamin E intake data from 387 participants were available from an externally validated Flower-Food Frequency Questionnaire (FFQ). The associations of plasma α- and γ-tocopherol, dietary vitamin E intake, with urinary α- and γ-CEHC were assessed using multivariate linear regressions.p = 0.02; std.beta: 0.06, p = 0.01, respectively). Furthermore, the sum of 24 h urinary α- and γ-CEHC excretions was positively associated with dietary vitamin E intake , whereas there was no relation between urinary α- and γ-CEHC/creatinine ratios and vitamin E intake. No association was observed neither between plasma α- and γ-tocopherol and dietary vitamin E intake, nor between urinary γ-CEHC and plasma γ-tocopherol.24 h Urinary excretion of α-CEHC (median (IQR): 0.9 (0.3–2.4) µmol) was less than that of γ-CEHC (median (IQR): 1.5 (0.5–3.5) µmol). After adjustment for covariates, we found that 24 h urinary α-CEHC excretion and urinary α-CEHC/creatinine ratio were both positively associated with plasma α-tocopherol (std.beta: 0.06, Our study confirmed our hypothesis that 24 h urinary α- and γ-CEHC excretions would be a better marker for dietary vitamin E intake than urinary α- and γ-CEHC/creatinine ratios. Considering that both 24 h urinary α- and γ-CEHC excretions and α- and γ-CEHC/creatinine ratios were also associated with plasma α-tocopherol status, we suggest that 24 h urinary α- and γ-CEHC excretions could be used to assess overall vitamin E status.The online version contains supplementary material available at 10.1007/s00394-022-02918-8. Vitamin E is the generic term for a group of lipid-soluble molecules consisting of four tocopherols  and four tocotrienols . Major dietary sources of vitamin E are nuts and edible vegetable oils, and the proportions of the four tocopherols vary according to the food source, with α-tocopherol and γ-tocopherol as most abundant representatives , 2.Although plasma α-tocopherol has been widely used to assess vitamin E status , there ir = 0.29, p = 0.01) . Serum α-tocopherol inadequacy has been set at < 30 μmol/L [24 h Urine samples were collected at baselines and stored at -80 °C until analysis. Prior to extraction of vitamin E metabolites α- and γ-CEHC (Supplementary figure S2), urine samples were enzymatically treated with beta-glucuronidase . Therefore, to 200 µL urine, 12.5 µL ascorbic acid (0.1 mg/mL) and 25 µL beta-glucuronidase (3 mg per 100 µL 0.1 M acetate buffer) were added and incubated for 2 h at 37 °C and 300 rpm. After enzyme treatment, samples were cooled on ice and acidified with 50 µL acetic acid. Extraction was performed twice with 1 mL hexane:dichloromethane (50:50 containing 0.1% BHT). Organic upper layers were combined and evaporated to dryness under vacuum. Samples were re-suspended in 50 µL acetonitrile:water (10:90) and 40 µL were injected into the high-performance liquid chromatography (HPLC) system. The separation was performed on a C18 column  and a Jasco HPLC system consisting of a AS-2059 Plus autosampler, PU-2080 Plus pump, and LG-2080–02 gradient unit; a three-line degasser  equipped with an electrochemical detector SA Model 5600A CoulArray Detector . The electrode potentials were set to 250, 325, 400, and 475 mV. The mobile phase consisted of 50 mM ammonium acetate and acetonitrile (80:20), and the flow rate was set to 1.5 mL/min. The column and electrodes were maintained at 40 °C and the autosampler cooled to 4 °C. Quantification was carried out against external authentic standard curves  (Supplementary figure S3). The method for the quantification of α- and γ-CEHC was precise (≤ 6.8%CV at high (20 µmol/L) and ≤ 9.1%CV at low concentrations (0.1 µmol/L)), accurate (≤ 3.6%bias at high and < 18.4%bias at low concentrations), and a recovery of 96.4% at high and 108.3% at low concentrations of α-CEHC and 99.2% at high and 108.3% at low concentrations of γ-CEHC. The lower limit of quantification (LLOQ) was 0.9 nmol/L for α-CEHC and 0.1 nmol/L for γ-CEHC. Urinary α- and γ-CEHC concentrations were presented as µmol/L and normalized for urinary creatinine as µmol/mmol creatinine, and subsequently, the 24 h urinary α- and γ-CEHC excretions were calculated by multiplying urinary α- and γ-CEHC concentrations with the recorded 24 h urine volume.Habitual dietary fat and energy intakes were estimated from a semi-quantitative self-reported food frequency questionnaire (FFQ) using the 2011 Dutch food composition database (NEVO) . The FFQt}× 0.01 . Howevert}× 0.01 , because2). The BMI was additionally categorized into underweight (BMI < 20 kg/m2), normal (20 ≤ BMI < 30 kg/m2), and overweight and obese (BMI ≥ 30 kg/m2) according to the fact sheet provided for older adults by the Netherlands nutrition center [2) = Anthropometric measurements and blood pressure (BP) were measured by well-trained staff. Anthropometric measurements were taken without shoes. Body weight was measured to 0.1 kg on a SECA 761 scale ; height was measured to 0.5 cm using the Frankfort Plane position by the SECA 222 stadiometer  . Body man center . Body surameters .Smoking status and vitamin supplement use were derived from self-administrated questionnaires. Smoking status was categorized into never, former, and current smoker. Supplementation use was categorized binarily into the use of any vitamin supplements or none. The use of lipid-lowering medication was included as a binary variable reported from participants and subsequently recoded into the anatomical therapeutic chemical (ACT) code (C10A and C10B), as lipid-lowering medication may directly impact plasma tocopherol status .p values of the correlation coefficient were adjusted for multiple tests.Participants’ characteristics were presented for all, and continuous data are presented as the mean ± standard deviation (SD) when normally distributed or as the median and interquartile range (IQR) for non-normally distributed variables. Categorical variables are presented as percentages. Between-group differences were assessed by ANOVA, Kruskal–Wallis, and Chi-square two-sided tests for normally distributed continuous data, non-normally distributed continuous data, and categorical data, respectively. Pairwise Pearson correlations were computed among 24 h urinary α- and γ-CEHC excretions, the sum of 24 h urinary α- and γ-CEHC excretions, urinary α- and γ-CEHC/creatinine ratios, plasma α- and γ-tocopherol, and dietary vitamin E intake. The The cubic roots of 24 h urinary α- and γ-CEHC excretions and urinary α- and γ-CEHC/creatinine ratios, and log-transformed dietary vitamin E intake were used in the regression models to increase the normality. In addition, plasma α- and γ-tocopherol were normalized for plasma total lipids by dividing tocopherol concentrations by the total lipids. We applied non-linearity test using the R-package “rms” to assess the relationship between urinary vitamin E metabolites, plasma tocopherols, and dietary vitamin E intake. Since we did not observe any non-linearity among them, linear regression and logistic regression models were built to assess those relationships. All linear regression models were presented as standard coefficient (95% CI).First, several linear regression models were built to assess the associations of 24 h urinary α- and γ-CEHC excretions, the sum of α- and γ-CEHC urinary excretions, and urinary α- and γ-CEHC/creatinine ratios with plasma α-tocopherol and γ-tocopherol, respectively. Univariate and multivariate models adjusted for age, sex, BSA, TC, 25-hydroxyvitamin D, use of lipid-lowering medication, smoking status, and SES were shown. Second, linear regression models were built to assess the associations of 24 h urinary α- and γ-CEHC excretions, the sum of α- and γ-CEHC urinary excretions, and urinary α- and γ-CEHC/creatinine ratios with dietary vitamin E intake. Univariate and multivariate models adjusted for age, sex, BSA, supplementation use, dietary lipids intake, smoking status, and SES were shown. Then, we assessed the relationships of plasma α- and γ-tocopherol with dietary vitamin E intake also by using univariate model and multivariate models adjusted for age, sex, BSA, supplementation use, dietary lipids intake, smoking status, and SES.Potential interactions were also explored by fitting interaction terms between urinary α- and γ-CEHC/creatinine ratios, 24 h urinary α- and γ-CEHC excretions as well as by age, sex, BSA, smoking status, SES, and 25-hydroxyvitamin D with each vitamin E urinary metabolites in associations with plasma α- and γ-tocopherol or vitamin E intake. Missing information on covariates in the association between CEHCs and tocopherols  was imputed using ten folded multiple imputation using the mice package in R.All analyses were carried out using RStudio .Participants’ characteristics are shown for all in Table p < 0.01 for both). Moreover, plasma α-tocopherol level was positively corelated with urinary α- and γ-CEHC in both molar creatinine ratio and 24 h excretion forms, with adjusted correlation coefficients between 0.06 and 0.08 (p < 0.05) (Supplementary table S1).Pearson correlation tests showed that dietary vitamin E intake was positively correlated with 24 h urinary excretion of α-CEHC and the sum of 24 h urinary excretions of α- and γ-CEHC, with adjusted correlation coefficients of 0.1 and 0.13 , p = 0.007, respectively), but not with plasma γ-tocopherol (Table Table p = 0.05) (Table p = 0.03) : 0.07 (0.01–0.15), 5) Table . More imIn this study, we aimed to explore the relationship of urinary vitamin E metabolites, i.e., α- and γ-CEHC, represented in both 24 h urinary excretions and molar creatinine ratios with plasma tocopherols and dietary vitamin E intake. We found that both 24 h urinary α-CEHC excretion and urinary α-CEHC/creatinine ratio were positively associated with plasma α-tocopherol status, and the sum of 24 h urinary α- and γ-CEHC excretions was positively associated with dietary vitamin E intake, which was in agreement with our hypothesis that 24 h urinary excretions of α- and γ-CEHC might be a better marker for vitamin E intake than urinary α- and γ-CEHC/creatinine ratios.. reported a strong adjusted correlation of urinary α-CEHC with plasma α-tocopherol [We included a rather large population to investigate the relation between plasma tocopherols and urinary excretions of CEHC, and found that both 24 h urinary α-CEHC excretion and urinary α-CEHC/creatinine ratio could be an indicator of plasma α-tocopherol status. Assessing plasma α-tocopherol status is of clinical importance, because circulating plasma α-tocopherol has been associated with metabolic syndrome and gallstone disease , 28. Ourcopherol , we failcopherol . A studycopherol , less fucopherol , and thecopherol –34.In the last 2 decades, the urinary vitamin E metabolite α-CEHC has been suggested to be a novel indicator of an adequate vitamin E intake . The phyThe dietary vitamin E intake assessed in our study included all dietary tocopherols, using a specially designed and externally validated “Flower-FFQ” instead of relying solely on α-tocopherol, which explains why we did not observe statistically significant association between 24 h urinary excretion of α-CEHC or γ-CEHC alone with dietary vitamin E intake in our multivariate models. More specifically, the sum of 24 h urinary excretion of α- and γ-CEHC would better correspond to the dietary vitamin E intake measured in our study, since α- and γ-tocopherols are the two most abundant tocopherols present in plants and in our diet. Thus, it was not surprising that we observed the positively association between the sum of α- and γ-CEHC, not α-CEHC or γ-CEHC individually, and dietary vitamin E intake. Although previous studies have mostly used urinary α- and γ-CEHC concentrations or α- and γ-CEHC normalized for urinary creatinine, due to the unavailability of 24 h urine, some studies have shown that urinary α-CEHC excretion responds to α-tocopherol supplementation in healthy men or/and women , 10, 35 Our findings support the current opinion that urinary excretion of CEHC has the potential to be a novel biomarker of dietary vitamin E intake, and meanwhile add evidence in a general population setting for future policymakers to make decisions on the requirements of vitamin E at a population level. In Europe, the current dietary reference values for vitamin E are set as Adequate Intake (AI) for α-tocopherol for all population groups based on observed intakes estimated from dietary surveys in healthy populations with no apparent α-tocopherol deficiency . UnfortuThis study has several strengths. First, we innovatively incorporated a new angle of urinary CEHC excretion and compared the utilization of both 24 h urinary excretions of CEHC and urinary CEHC/creatinine ratios regarding assessing vitamin E intake and status. Second, we assessed the 24 h urinary excretions of α- and γ-CEHC and plasma tocopherols in a relatively large population, compared to relevant literature, which could increase the generalization of our results. Third, the vitamin E intake was assessed by an externally validated and tailored FFQ designed for the Lifelines Cohort Study, and thus, to some extend increased the accuracy and reliability of vitamin E intake data . HoweverIn conclusion, our findings highlight the importance of measuring urinary vitamin E metabolites by showing that 24 h urinary α-CEHC excretion could be an indicator of plasma α-tocopherol status, and the sum of 24 h urinary excretions of α- and γ-CEHC could better assess dietary vitamin E intake compared to urinary α- and γ-CEHC/creatinine ratios. More research in a general younger population is needed to pool sufficient evidence for validating 24 h urinary excretions of α- and γ-CEHC as valid biomarkers for vitamin E intake and status.Supplementary file1 (DOCX 268 KB)Below is the link to the electronic supplementary material.