According to Nuclear Magnetic Resonance (NMR) spectroscopy, single crystal X-ray analysis and Density Functional Theory (DFT) calculations, the trop‒ and trop2−• anions show anti-aromatic properties which are dependent on the counter cation M+ and solvent molecules serving as co-ligands. For comparison, the disodium and dipotassium salt of the dianion of dibenzocyclooctatetraene (C16H12 = dbcot) were prepared, which show classical aromatic character. A d8-Rh(I) complex of trop− was prepared and the structure shows a distortion of the C15H11 ligand into a conjugated 10π -benzo pentadienide unit—to which the Rh(I) center is coordinated—and an aromatic 6π electron benzo group which is non-coordinated. Electron transfer reactions between neutral and anionic trop and dbcot species show that the anti-aromatic compounds obtained from trop are significantly stronger reductants.The synthesis, isolation and full characterization of ion pairs between alkaline metal ions (Li Based on the 4n-Hückel-rule, the trop anion, C15H11−, with its central 8π electron system flanked by two annulated benzo groups , should show anti-aromatic character. Indeed, when the trop anion is generated in situ in liquid ammonia by deprotonation of the neutral hydrocarbon suberene (tropH) ((a) in 1H NMR spectrum, as expected for an anti-aromatic compound cyclooctatetraene, dbcot, with alternating single and double bonds  in Mdbcot containing the dbcot−• anion radical and then to M2dbcot with the dbcot dianion (vide infra) [Mdbcot suggest that there are contacts between the cations M+  and dbcot−• [2[dbcot] (tmeda = tetramethylethylenediamine) was determined. In this ion triple, the dianion adopts a planar structure with nearly equal C-C bond lengths in the central ring  Å) . Reductie infra) ,29. The  dbcot−• ,30. Only dianion . The 1H in dbcot  to 7.08 Li2dbcot . In combaromatic .2trop which allows the isolation of various close and separated ion pairs which contain the trop mono- and di-anion. For comparison, the disodium and dipotassium salts of dbcot2− were also prepared and structurally characterized. Their anti-aromatic or aromatic character is assessed experimentally based on structural and magnetic criteria. DFT-calculations on simplified model compounds, specifically calculation of the Nuclear Independent Chemical Shifts (NICS), were performed in order to bolster the conclusions derived from the experimental data.Herein, we report a new simple and clean synthesis of Mtrop and M– in pure form, the silylated trop derivatives 1a (R = H) cyclooctatetraene (dbcot) to elementary sodium or potassium graphite in anhydrous tetrahydrofuran ((c) in 2dbcotLi  gives [Rh(trop)(cod)] as a dark red crystalline compound in moderate isolated yield (63%) ((d) in 8-valence electron configured Rh(I) center is exclusively coordinated to hydrocarbon ligands and represents to the best of our knowledge the first fully characterized mononuclear Rh(I) heptatrienide species. Only very few related compounds such as bimetallic Rh complexes  were grown and investigated with X-ray diffraction methods in order to determine the structures experimentally. In addition, the structures of compounds Mtrop (M = Li − K) and [Rh(trop)(cod)] and their degree of anti-aromatic or aromatic character was evaluated by calculating the Nuclear Independent Chemical Shifts (NICSs) .‒ anion show strongly shielded signals for all protons in the 1H NMR spectra. In the dimer of the trop radical + cation binds predominantly to C1 [Li-C1 2.279(3) Å] which provokes a rather strong bending , about half of the one observed in the hydrocarbon trop2 . Encapsulation of Li+ by three dimethoxyethane (dme) solvent molecules leads to [Li (dme)3]+[trop−] (dmetropLi), in which no close contact between cation and anion occurs (shortest Li–C contact = 6.79 Å) and the trop‒ anion adopts an almost flat structure . Likewise, in the structure of Natrop, which contains [Na(dme)3]+, the contact between cation and anion is long (the distance between Na+ and the centroid (cnt) of the central C7 ring is 5.25 Å) and the trop‒ anion is flat .The experimental structures of the contact ions pairs 1 and Θ2 . These a‒ anion is observed in thftropK , which is best described as a close ion pair with a rather short distance between K1 and the centroid cnt1 of the C7 ring of 2.82 Å. The K+ is coordinated to the C7 ring in a slightly asymmetric manner as indicated by the unequal K-C1 (3.098 Å) and K-C4/C5 distances . Furthermore, the potassium ion has an additional contact at about 2.97 Å to another trop‒ unit, such that K+…trop−…K+….trop− chains are formed in the solid state. Remarkably, in 18c6tropK, the trop− anion shows larger fold angles . In this ion pair, K+ is rather asymmetrically bound and the distance to the centroid of to the central C7 ring is large (3.812 Å). Instead, K+ binds closer to the allylic fragment C1, C7, C15 (K1-cnt2 3.303 Å) with a rather short contact to the benzylic carbon center C1 (3.02 Å) and longer ones to C7 (3.263 Å) and C15 (3.490 Å). It is highly speculative to which extent these features of the solid-state structures are retained in solution. But, the coupling constants 1JHC of the benzylic carbon nucleus, 13Cbz, to the benzylic proton, 1Hbz, can be empirically correlated to the s-orbital character in the Cbz–H bond. In a bent trop anion, the Cbz–H bond will be closer to a sp3 configuration  that in a flat structure, where the Cbz–H is closer to a sp2 configuration . Indeed, 1JHC obtained in deuterated tetrahydrofuran, [D8]THF, as solvent (cod)]. In contrast to Mtrop, which shows strongly shielded 1H NMR resonances for all protons, the signals for the 1H nuclei at the benzo groups in [Rh(trop)(cod)] are in the same region (δ 1H: 6.82–7.01 ppm) as in the reference compound 2trop (δ 1H: 6.58–7.25 ppm). Also, the chemical shifts of the benzo 13C nuclei are in the normal range for arenes in between δ = 127 and 137 ppm. Only the olefinic and benzylic 1H and 13C NMR signals  are significantly shifted to lower frequencies, indicating an interaction with the metal center, c.f. δ (13Col) = 97.6 ppm, δ (13Cbz) = 56.5 ppm in [Rh(trop)(cod)] vs. δ (13Col) ~139 ppm and δ (13Cbz) = 81.6–89.4 ppm in Mtrop, see [Rh(trop)(cod)] is seemingly 2vC-symmetric which is not in agreement with the structure in solid state but can be explained by a dynamic phenomenon (vide infra). Complex [Rh(trop)(cod)] crystallizes as racemic mixture in the space group P21/c. The structure of one of the enantiomers is depicted in Mtrop compounds. The Rh(I)(cod) fragment binds to C1, C2, C3, C4 and C5 at distances between 2.09 to 2.40 Å . The most notable structural feature is the strongly bent conformation of the trop‒ unit: the intersection angle between the non-coordinated benzo group and the benzopentadienide unit is θ3 = 49.9(1)° (cod)] underlies a dynamic phenomenon by which the rhodium center rapidly exchanges between the two possible 5η binding sites of the central seven-membered ring, as shown in a,d]cycloheptenide metal complex C  = 70–100 ppm]. That is, in this case, the anti-aromatic π-electron system of trop‒ is localized in a different way from the one in [Rh(trop)(cod)], namely into a 6π-electron system at a terminal arene bound to the metal and an uncoordinated 10π-electron system.At this point, it is interesting to compare these main group metal complexes of trop(1)° cf. . The η5 omplex C d 25,26],26− with2tropK, 2dbcotNa and 2dbcotK with the trop2‒• dianion radical or the dbcot2‒ dianion respectively, are shown in The structures of the ion triples Ktrop to the radical dianion 2tropK leads to an overall elongation of all bonds within the central seven-membered ring  as solvent, the EPR spectrum of 2tropK shows a rather complex hyperfine coupling pattern . The less complex spectrum  show, in comparison to neutral dbcot, which is best described as a cyclic conjugated polyolefin, a diatopic ring current which leads to significantly de-shielded olefinic resonances, 1Hol > 7 ppm  is listed for comparison which is considered to be an archetypical anti-aromatic molecule with a 4-electron system and shows positive NICS values ((NICS(0) = 27.49, NICS(1) = 18.03). The values are largely exceeded by the NICS values of the central ring B in the flat and counter cation-free trop anion ‒trop which amount to 45.00 and 34.54. Even the flanking rings A and C in ‒trop reach positive values in the range of CBD. For simplicity, the NICS data were calculated for simplified model compounds of the contact ion pairs mMtrop  and 2tropmK, where the solvent molecules bound to M+ were not considered. Nevertheless, the calculated π–π* transitions of the calculated UV/Vis spectra correspond well to the recorded UV/Vis spectra , the smaller the NICS values are and the less pronounced the anti-aromatic character. On the contrary, when the bending becomes small such as in Ktropm, the NICS values reach the ones of flat ‒trop. While it may be debated whether CBD is an anti-aromatic compound because of its rectangular distortion  are negative and indicate modest magnetic aromatic character.As already stated above, the trop2‒• are much smaller than for the mono-anions and indicate diminished anti-aromatic character in accord with conclusions derived from a comparison of the experimental structural data  are listed in For completeness, the NICS(0) and NICS(1) data for the free dianion 2trop, Ktrop and dbcot were investigated to provide some understanding of the reactivity, especially with respect to mutual electron exchange processes. As previously reported, the cyclic voltammogram of dbcot shows two close lying reduction events at E° = −2.24 V and E° = −2.33 V vs. Fc/Fc+, resulting from two consecutive one-electron transfer steps to give a planar dbcot2− dianion  3‒ ). The dereaction . In addireaction . This be. Fc/Fc+  are obsetion see . In contechanism . The rev2trop in the presence of 10 equivalents of KPF6 showed anodically shifted redox waves, indicating that potassium ions contribute in stabilizing the trop anions and in facilitating the C–C bond cleavage of trop2  in 6 oxidizes Ktrop to reform 2trop (step (b) in Ktrop is further reduced to 2tropK with potassium graphite (see (c) in 2tropK is a strong reductant (E° = –3.04 V). Addition of half an equivalent 2trop oxidizes 2tropK to give the mono-anion Ktrop, although heating to 60 °C is required ((d) in dbcot to 2tropK, the former is fully reduced to the aromatic dianion 2dbcotK ((e) in The electrochemical behavior of dbcot is used in the oxidation of the dianion radical 2‒•trop, a mixture of 2Kdbcot , aside 0.64 equiv. Ktrop and 0.16 equiv. of 2trop is obtained according to 1H NMR spectroscopy. A featureless EPR signal (g = 2.00485) suggests that also K+[dbcot‒•] may be present [+[dbcot‒•] (≈0.06 equiv.) is in a rapid equilibrium with the remaining neutral dbcot molecules (≈0.3 equiv.) which we could not detect by 1H NMR spectroscopy. We also reacted two equivalents of the anti-aromatic mono-anionic ion pair Ktrop with dbcot. Also, in this reaction, a product mixture consisting of 2Kdbcot (0.5 equiv.), unreacted Ktrop (0.9 equiv.) and 2trop (0.5 equiv.) is obtained. EPR spectroscopy indicates that, very likely, K+[dbcot‒•] (g = 2.00565) is formed in this reaction as well  as a major product (95%) and not as expected Ktrop or 2trop, which is formed in only 5% yield ((f) in 2dbcotK, which contains the diamagnetic and aromatic dbcot2‒ dianion, is very clean and gives the neutral hydrocarbon dbcot in almost quantitative yield ((g) in Finally, the oxidation of the dianion salts 8 [2Cl2(cod)2]  . THF, DMocarbons  52]. Fu. Fu8 cycloheptene  and elementary lithium  were reacted in dry tetrahydrofuran (40 mL) at a.T. overnight. The resulting dark red liquid was added dropwise to a solution of chlorodimethylsilane  in dry tetrahydrofuran (40 mL) over an ice bath. The resulting yellow liquid was allowed to warm to a.T. and stirred overnight. The turbid suspension was concentrated under reduced pressure and subsequently distilled in high vacuum (only one fraction was observed from 90 to 115 °C). The obtained yellow liquid was placed in the fridge to give a white, crystalline solid after three days, which was triturated with dry diethyl ether (10.0 mL) and dried in high vacuum .trop–Si(H)Me1H NMR  δ 7.27–7.09 , 7.06 , 6.74 , 4.18–4.07 , 3.49 , −0.09  ppm. 13C NMR  δ 140.83 , 135.05 , 132.59 , 129.59 , 129.13 , 128.96 , 125.71, 47.60 , −3.71 2) ppm. ATR IR: λ−1: 3015 , 2955 , 2143 , 868 (s), 796 (s), 726 (s), 446 (s) cm−1. Elemental analysis: C ; H .3 (1b): 5-Chloro-5H-dibenzocycloheptene  and elementary lithium  were reacted in dry tetrahydrofuran (30 mL) at a.T. overnight. The resulting deep red liquid was added dropwise to a colorless solution of trimethylsilyl chloride  in dry tetrahydrofuran (35 mL). The reaction mixture was allowed to warm to a.T. and stirred overnight. The solvent of the orange solution was removed under reduced pressure to give an orange solid. The crude product was purified by sublimation  to give a yellow, crystalline solid .trop–SiMe1H NMR  δ 7.08 , 7.06–6.92 , 6.86 , 6.48 , 3.41 , −0.06  ppm. 13C NMR  δ 140.91 , 135.49 , 132.86 , 129.95 , 129.73 , 129.07 , 125.71 , 50.24 , −0.39  ppm. Elemental analysis: C ; H .Litrop: Method A: Lithium sand  was suspended in dry tetrahydrofuran (10 mL) and placed in a cooling bath (propan-2-ol and dry ice). 5-Chloro-5H-dibenzocyclo-heptene  was dissolved in dry tetrahydrofuran (30 mL). The resulting light yellow solution was added dropwise (during 25 min) to the brown suspension. The reaction mixture was allowed to warm to a.T. and a color change to dark red was observed under heat development. After two hours, the dark red liquid was concentrated to approximately 25 mL and filtered over a plug of celite. The resulting red solution was layered with dry n-hexane (71 mL) and stored in the freezer . A first crop of green, crystalline needles was obtained . The supernatant was layered with dry n-hexane (30 mL) and stored in the freezer  and a second crop was obtained .H-Dibenzocycloheptene-5-yl)-dimethyl-silane  was dissolved in dry 1,2-dimethoxyethane (2.0 mL). The resulting slightly yellow solution was added to a white suspension of lithium tert-butoxide  in dry 1,2-dimethoxyethane (2.0 mL) (in a solvent with lower polarity (such as diethyl ether or tetrahydrofuran), a significantly lower yield was obtained). A swift color change to dark red was observed and the reaction mixture was stirred at a.T. (3 h). The dark red liquid was filtered over glass filter paper and celite, layered with dry n-hexane (10.0 mL) and stored in the freezer . The light yellow supernatant was discarded and the obtained green crystals were washed with dry n-hexane (3 × 1 mL) and dried under a stream of argon .Method B:  δ 5.70 , 5.15 , 4.87 , 4.59 , 3.92 , 1.54  ppm. 1H NMR  δ 4.80–4.73 , 4.12–4.05 , 3.73–3.66 , 3.48–3.42 , 2.64 , 0.50  ppm. 13C NMR  δ 163.60 , 139.03 , 138.27 , 132.65 , 132.05 , 120.67 , 112.44 , 83.67  ppm. UV/Vis (THF): λmax 233 (ε: 1276(321) m2mol−1), 287 (ε: 2984(340) m2mol−1), 443 (ε: 631(102) m2mol−1), 513  m2mol−1) nm. ATR IR: λ−1: 2985 , 2926 , 1364 (s), 1068 (s), 912 (s), 724 (s), 436 (s) cm−1. Elemental Analysis for Litrop(dme)1.7: C ; H . MP: 62 °C (decomp.).Natrop: -dimethyl-silane  was dissolved in dry diethyl ether (30 mL). The resulting colorless solution was added to a white suspension of sodium tert-butoxide  in dry diethyl ether (60 mL) over an ice bath. After 30 min, the reaction mixture was allowed to warm to a.T. and a dark red liquid was obtained. The solvent was removed under reduced pressure and dry 1,2-dimethoxyethane (30 mL) was added to the red residue. The liquid was filtered over a plug of celite, layered with dry n-hexane (80 mL) and stored in the freezer . The light yellow supernatant was discarded and the obtained dark green crystals were washed with dry n-hexane (2 × 10 mL) and briefly dried in vacuo .1H NMR  δ 4.76 , 4.08 , 3.68 , 3.43 , 2.60 , 0.56  ppm. 1H NMR  δ 5.18 , 4.54 , 4.11 , 3.93 , 2.98 , 1.10  ppm. 13C NMR  δ 161.95 , 138.79 , 136.39 , 133.16 , 132.57 , 120.12 , 113.22 , 81.56  ppm. UV/Vis (THF): λmax 285 (ε: 3074(349) m2mol−1), 459 (ε: 803(158) m2mol−1) nm. ATR IR: λ−1: 3039 , 3003 , 2978 , 2932 , 2913 , 2869 , 2823 , 1553 (s), 1443 (s), 1362 (s), 1118 (s), 1082 (s), 1028 (s), 734 (s) cm−1. Elemental Analysis for Natrop(dme)1.1: C ; H . MP: 45 °C (decomp.).§ The upper part of the multiplet lies underneath the signal of the solvent DME.Ktrop: Method A: -dimethyl-silane  and potassium tert-butoxide  were placed under argon. Dry tetrahydrofuran (20 mL) was added and a dark red liquid was obtained immediately. The reaction mixture was stirred at a.T. (10 hr) and filtered over a plug of celite. The dark red filtrate was layered with dry n-hexane (68 mL) and stored in the freezer . A first crop of red crystals was isolated . The supernatant was layered with dry n-hexane (60 mL), stored in the freezer  and a second crop was obtained .H-Dibenzocycloheptene-5-yl)-trimethyl-silane  and potassium tert-butoxide  were placed under argon. Dry tetrahydrofuran (35 mL) was added over an ice bath. The yellow solution was allowed to warm to a.T. and a gradual color change to dark red was observed. The reaction mixture was stirred overnight. The solvent was removed under reduced pressure. The red residue was triturated with dry diethyl ether (2 × 20 mL) and then washed with dry diethyl ether (40 mL). The resulting red solid was briefly dried in vacuo . The red solid  was recrystallized in dry tetrahydrofuran (5.0 mL), filtered over glass filter paper and celite, layered with dry n-hexane (10.0 mL) and stored in the freezer . The long, crystalline needles were washed with dry n-hexane (4 × 1.5 mL) and dried under a stream of argon .Method B:  δ 4.47 , 3.74 , 3.26 , 2.97 , 2.00 , 0.06  ppm. 1H NMR  δ 4.40 , 3.65 , 3.18 , 2.89 , 1.91 , −0.01  ppm. 13C NMR  δ 162.96 , 139.26 , 138.42 , 133.95 , 132.89 , 120.17 , 111.87 , 89.44  ppm. UV/Vis (THF): λmax 286 (ε: 3343(566) m2mol−1), 296  m2mol−1), 478 (ε: 1025(277) m2mol−1) nm. ATR IR: λ−1: 3006 , 2985 , 2939 , 2924 , 1366 (m), 1121 (s), 1039 (m), 914 (m), 797 (m), 740 (s), 436 (m) cm−1. Elemental Analysis: C ; H . MP: 240 °C (decomp.).2tropK: Ktrop  was dissolved in dry tetrahydrofuran (5.0 mL). The resulting dark red solution was added to a golden suspension of potassium graphite  in dry tetrahydrofuran (5.0 mL) and stirred at a.T. (3 days). The dark red suspension was filtered over a plug of celite and the dark red filter cake was washed with dry tetrahydrofuran (3.5 mL). The dark red filtrate was split into two portions and each was layered with dry n-hexane (6.0 mL) and stored in the freezer . The dark red, microcrystalline solid of both fractions was washed with dry n-hexane (2 × 2 mL each) and briefly dried in vacuo . The supernatant of both fractions was concentrated under reduced pressure and the dark residue was recrystallized again from dry tetrahydrofuran (3.0 mL) and dry n-hexane (6.0 mL) at −30 °C to give a second crop of dark red, microcrystalline solid .max 286 (ε: 4098(190) m2mol−1), 297  m2mol−1), 361 (ε: 1988(237) m2mol−1), 482 (ε: 1633(37) m2mol−1) nm. ATR IR: λ−1: 3020 , 2970 , 2868 , 1324 (m), 1015 (m), 736 (m), 686 (m), 433 (m) cm−1. Elemental Analysis for K2trop(thf)(n-hexane)0.2: C ; H . MP: >250 °C.UV/Vis (THF): λ2dbcotNa: Dibenzocyclooctatetraene  was dissolved in dry tetrahydrofuran (10 mL). The colorless solution was added to a Schlenk tube with a sodium mirror . A swift color change to dark red was observed. The reaction mixture was stirred at a.T. (1 day). The solvent was removed under reduced pressure. The purple residue was suspended in dry tetrahydrofuran (7 mL) and filtered over a plug of celite to give a dark red filtrate (a) and a dark red filter cake (b). (a) The dark red filtrate was layered with dry n-hexane (10 mL) and stored at a.T. (7 days). The obtained dark crystals were washed with dry n-hexane (2 mL) and dried under a stream of argon . (b) The filter cake was extracted with dry 1,2-dimethoxyethane (6 mL). The dark red filtrate was layered with dry n-hexane (8 mL) and stored in the freezer . The obtained dark crystals were washed with dry n-hexane (2 × 2 mL) and dried under a stream of argon .1H NMR  δ 7.87 , 7.17 , 6.21  ppm. 13C NMR  δ 135.26 , 108.90 , 107.43 , 93.37  ppm. UV/Vis (THF): λmax 238 (ε: 2523(2) m2mol−1), 261  m2mol−1), 328 (ε: 5293(512) m2mol−1), 392 (ε: 853(87) m2mol−1), 514 (ε: 94(12) m2mol−1), 553 (ε: 106(13) m2mol−1), 597 (ε: 76(9) m2mol−1) nm.2dbcotK: Dibenzocyclooctatetraene  was dissolved in dry tetrahydrofuran (12 mL). Potassium graphite  was added. The resulting dark red suspension was allowed to stir at a.T. (1 day). The reaction mixture was filtered over a plug of celite. The filter cake was washed with dry tetrahydrofuran (3 mL). The filtrate was split into two portions and each layered with dry n-hexane (8 mL) and stored in the freezer . The obtained dark green crystals were washed with dry n-hexane (2 × 1 mL) and briefly dried in vacuo . The supernatant was concentrated under reduced pressure and the black residue was recrystallized in the same manner to give a second crop .1H NMR  δ 7.87 , 7.17 , 6.19  ppm. 13C NMR  δ 135.72 , 109.36 , 108.91 , 95.78  ppm. UV/Vis (THF): λmax 235 (ε: 3997(184) m2mol−1), 261  m2mol−1), 326 (ε: 1959(338) m2mol−1), 388 (ε: 334(53) m2mol−1), 502 (ε: 38(6) m2mol−1), 544 (ε: 42(7) m2mol−1), 587 (ε: 31(5) m2mol−1) nm. MP: >250 °C.[Rh(trop)(cod)]: Cyclooctadiene rhodium(I) chloride dimer  and Ktrop  were stirred in dry toluene (6 mL) at a.T. (12 hr). The dark red liquid was filtered over a plug of celite, layered with dry n-hexane (6 mL) and stored in the freezer . The obtained dark red crystals were washed with dry n-hexane (0.5 mL) and dried under a stream of argon .1H NMR  δ 7.01–6.88 , 6.85 , 5.16 , 4.09 , 3.59 , 2.88 , 2.21 , 1.76  ppm. 13C NMR  δ 137.43 , 132.63 , 128.06 , 126.86 , 126.66 , 126.13 , 97.52 , 91.23 , 70.69 , 55.84 , 35.22 , 29.91  ppm.§ The peak is buried under the signal for C6D6.1H NMR  δ 7.10–6.99 , 6.89–6.84 , 5.46 , 3.92 , 3.70 , 2.81 , 2.30 , 1.76  ppm. 13C NMR  δ 137.75 , 133.25 , 128.54 , 127.42 , 127.24 , 126.78 , 97.57 , 90.61 , 70.35 , 56.45 , 35.09 , 30.46  ppm.tBu allowed the very clean synthesis of larger amounts of the salts of the trop anion, ‒trop, which contain alkali metal counter cations M+ = Li, Na, K. With these at hand, an in-depth study on the electronic configuration of these conjugated hydrocarbons exclusively composed from 2sp-valence electron hybridized carbon centers could be performed. As the Hückel counting rules imply, the 16π-electron configuration of trop‒ predicts this to be anti-aromatic. This is indeed the case as NMR data and as well as calculated NICS data clearly show. A number of contact ion pairs, Mtrop, could be structurally characterized by X-ray diffraction methods. In these, ethereal molecules complete the coordination sphere around the M+ cation. From tetrahydrofuran (THF), close ion pairs are obtained which show an intimate contact between cation and trop‒ anion. In these, the trop‒ anion is bent and its anti-aromaticity lowered. On the contrast, crystallization from solvents, which have a higher solvation energy for alkali cations, lead to separated ion pairs in which the trop‒ anion is flatter and consequently, more anti-aromatic. Moreover, Li+ shows a higher tendency to form contact ion pairs than the larger potassium cation with a significantly reduced charge density. This culminates in the isolation of [K(thf)2][trop]∞, which forms a one-dimensional coordination polymer in the solid state with long K+ trop‒ distances. This compound contains a flat trop‒ anion which shows very little variation of the C=C bond lengths, strongly shielded NMR signals, strongly positive NICS values and as such, fulfils all formal criteria requested for an anti-aromatic compound. In this light, the ease of synthesis from trop silyl ethers and alcoholates is somewhat surprising, indicating that Mtrop salts are remarkably stable. The trop‒ monoanion can be further reduced to give salts 2tropM with a paramagnetic dianion radical, trop2‒•, which was isolated and fully characterized as 2tropK. In this species, the anti-aromaticity is reduced compared to the mono-anion. In a reaction between a Rh(I) halide complex and trop‒, the complex [Rh(trop)(cod)] was prepared. Not unexpected, this pure organometallic 18 electron complex shows a very different structure in which the trop unit—especially its annulated benzo groups—is aromatized. This reaction also shows that trop anions may have potential as ligands for transition metal complexes and as a building block for main group element compounds. It remains to be seen in how far trop-type anions can be used as electron carriers related to the well-established use of arenes for that purpose [A desilylation reaction using silyl trop derivative which proceeds by addition of KO purpose .


EOs had varying compositions of mono- and sesquiterpenes determined by gas chromatography-mass spectrometry (GC-MS) analysis. The in silico molecular interactions screening of major EO constituents and the seven selected target proteins of the nematode indicated highest binding affinity of geraniol-ODR1 (odorant response gene 1) complex , due to extensive H-bonding, hydrophobic and π-alkyl interactions. The relative binding affinity followed the order: geraniol-ODR1 > β-terpineol-ODR1 > citronellal-ODR1 > l-limonene-ODR1 > γ-terpinene-ODR1. Taken together, the cumulative in vitro and computational bioefficacy analysis related to the chemoprofiles of EOs provides useful leads on harnessing the potential of EOs as bionematicides. The insight on biochemical ligand–target protein interactions described in the present work will be helpful in logical selection of biomolecules and essential oils for development of practically viable bionematicidal products.Nematicidal potential of essential oils (EOs) has been widely reported. Terpenoids present in most of the essential oils have been reported responsible for their bioactivity though very less is known about their modes of action. In the present study, an  Meloidogyne incognita is most abundant in tropical soils, barely sparing any crop family, and the most challenging part is to control its population below economic damage levels  are complex mixtures of terpenoids and their oxygenated derivatives, produced by isoprenoid pathways . Only ∼1ncognita . EOs of tivities . EO fromma index . Similarntration . Another μl mL–1 . The EO to plant . Promisiin vitro against M. incognita, and subject the most effective EOs to in silico analysis for a likely mode of action, using molecular docking and modeling approach.A review of literature clearly showed that EO bioactivity evaluation against nematodes largely remained restricted so far to the evaluation of EOs against different plant parasitic nematodes. Emphasis on the correlation of their anti-nemic activity with chemical compositions and mechanism of interaction at molecular level with the possible target sites of action has remained lacking. Therefore, the present study was performed to characterize the chemical composition of the selected EOs, evaluate their bio-efficacy Citrus sinensis (L.) Osbeck; family Rutaceae, orange essential oil), MTEO , CEO , TEO , AEO , MREO , CNEO , WEO , and PEO  were purchased from CDH Fine Chemicals  and Merck®,  and used without further purification.Commercially available EOs (99% purity) of different plants, namely, OEO . Surfactants, Atlas G5002 and Triton X-100 were procured from Croda India Company Pvt. Ltd.  and Loba Chemie Pvt., Ltd. , respectively. For GC-MS analysis, helium (He) gas of high purity (99%) was used.All the solvents used were of AR grade, purchased from Merck®, United States) equipped with an HP-5MS column  as stationary phase, which was directly connected to a triple axis HED-EM 5975C mass spectrometer . The injection volume was 1 μl with flow mode in split control. Helium was used as carrier gas at a head pressure of 10 psi, and flow was set at 1 ml min–1. The GC-MS condition was programmed with the oven temperature initially held at 40°C for 1 min, thereafter increased with a gradient of 3°C min–1, until the temperature reached to 120°C and held constant for 2 min. The temperature was raised again with a gradient of 5°C min–1 up to 220°C and held constant for 1 min and finally raised to 280°C with an increment of 4°C min–1. The total run time of the analysis was 65 min. The MS acquisition parameters were ion source temperature 180°C, electron ionization 70 eV, full scan mode (50–550 AMU), transfer line temperature 280°C, solvent delay 3 min, and E.M voltage 1,380 V. Compounds were identified by matching their mass spectra and fragmentation pattern using NIST  Mass Spectra Library. Further rentention indices (RI) have been calculated following Kovats (1978):Volatile constituents of EOs were analyzed in GC-MS in a 7890A GC instrument (Agilent Technologies∗ n + [log(RTcompound - v) - log(RT - v)]/[log - log]RI = 100 compound = the retention time of the compound, v = the column void time, RTlarger alkane = the retention time of the larger alkane, and RTsmaller alkane = the retention time of the smaller alkane.where n = the number of C in the smaller alkane, RT2s) of M. incognita were collected from roots of 21-day-old infected tomato seedlings. Nematode-infested soil was screened through water screening method following Cobb’s sieving and decanting technique (2s suspensions were combined and counted under light microscope.Nematode culture was maintained on infected tomato plants (var. Pusa Ruby) under greenhouse conditions. Second instar juveniles (Jechnique . Furtherechnique . Nematod–1) of all EOs except PEO were prepared using Atlas G5002 surfactant (2% w/w). In the case of PEO, Triton X-100 (2% w/w) was used. Each primary stock emulsion was diluted serially with surfactant solution to prepare secondary test emulsions of varying strengths .Primary stock emulsions , each containing EO emulsion (2 ml) of a particular test strength . Surfactant solutions used to dissolve EOs were taken as corresponding negative controls. Each treatment was replicated thrice. Multiwell plates were incubated at 27 ± 1°C and examined using a stereoscopic microscope at 24, 48, and 72-h intervals. The numbers of dead vs alive juveniles in each treatment was recorded. Motionless nematodes with straight bodies were counted. The revival test was done as described by 2s was calculated considering the mortality of juveniles in negative control.Nematicidal assay was conducted under ications . Treatmein vitro nematicidal assay and GC-MS analysis, major volatile constituents of OEO, CNEO, and TEO were selected out of nine EOs, for in silico ligand target protein interaction analysis.Based on the results of M. incognita were selected as target receptors for the molecular docking studies. Cytochrome c oxidase subunit 1 is involved in the oxidative phosphorylation pathway, which is part of the energy metabolism. AChE regulates synaptic transmission and locomotion processes. The full functional activity of Hsp90 is gained in coordination with other co-chaperones, playing an important role in the folding of newly synthesized proteins, stabilization and refolding of denatured proteins during stress. ODR1 and ODR3 regulate chemosensory functions. Neuropeptide GPCR is associated in the regulation of movement of the parasite toward (or within) its host. CLAVATA3/ESR (CLE)-related protein plays an important role in the differentiation or division of feeding cells (syncytia) induced in plant roots during infection. (Ref. for each).Seven target proteins, namely, cytochrome c oxidase subunit 1, AChE, Hsp90, ODR1, ODR3, neuropeptide GPCR, CLAVATA3/ESR (CLE)-related protein of 1,2 were used to search and annotate the molecular and biological functions of the query sequences. The NCBI Blast tool and the PDB database were together used to identify the templates for modeling the secondary structures of the query sequences. Further homology modeling of the proteins was carried out using Modeller v 9.24.The hypothetical protein sequences were taken from the NCBI and UNIPROT database . The BLA3. The ligand structures were minimized using MM2 forcefield in Chem Draw Ultra 11.0 software  and used for molecular modeling studies. The ligand molecules were customized for docking using the Dock prep tool of Autodock Vina. Hydrogen molecules were added, and the incomplete side chains were replaced using Dunbrack rotamer library .The bioassay experiments were done in triplicate. The significance of the differences between variables was tested using one-way ANOVA. The means were compared using Duncan’s multiple range test. Statistical significance was determined at l-limonene (93.2 ± 2.30%) was found to be most abundant in OEO, along with β-myrcene (2.2 ± 0.2%), α-pinene (1.6 ± 0.1%), sabinene (0.5 ± 0.1%), limonene oxide (0.2 ± 0.0%), and decanal (0.2 ± 0.0%). l-Limonene was confirmed based on its fragmentation pattern with characteristic daughter ion peaks of m/z 136.2, 108.2, and 71.2, generated due to sequential loss of methyl and ethyl moieties (γ-terpinene (17.5 ± 1.1%), α-terpinene (8.7 ± 0.6%), p-cymene (4.8 ± 0.2%), α-pinene (3.2 ± 0.2%), p-menth-8-en-2-ol (3.2 ± 0.5%), and 1,8-cineol (3.1 ± 0.4%). Besides, α-gurjunene (2.1 ± 0.1%) and δ-cadinene (1.6 ± 0.2%) were identified as the major sesquiterpenes.The compositions of EOs of OEO, MTEO, CEO, TEO, AEO, MREO, CNEO, WEO, and PEO were determined by comparing their mass spectra with data library, corresponding retention indices, and mass fragmentation patterns. The identified chemical constituents of the oils are listed in moieties . Interesl-limonene (5.7 ± 0.5%), β-elemene (3.3 ± 0.1%), and neryl acetate (2.1 ± 0.2%).Analysis of volatiles of CEO and CNEO revealed the presence of various terpenes, representing 93.5% and 95.8% of the total oil composition, respectively. These oils were characterized by the presence of predominant acyclic monoterpene aldehyde and citronellal with its respective contents of 81.9 ± 1.1% and 31.5 ± 1,1%, in two oils. Both CEO and CNEO showed higher content of oxygenated compounds, in which the former attributed an appreciably higher content primarily of 91.3% oxygenated monoterpenes. Similarly, CNEO mainly contained oxygenated terpenoids (81.9%) and hydrocarbons (13.9%). Except citronellal, other constituents of CEO were citronellol (5.8 ± 0.7%) and fenchone (2.3 ± 0.3%), while CNEO contained geraniol (30.6 ± 1.3%), citronellol (9.6 ± 0.3%), γ-muurolene (1.2 ± 0.2%) were also detected. Sesquiterpene content was found relatively higher in AEO (3.7%), whereas monoterpene content was meager (0.4%). Volatile composition of MREO showed abundance of furanoeudesm (41.9%) and curcerene (23.9%), considered as marker components of MREO. Besides these, other sesquiterpenoids such as β-elemene (4.8%), δ-elemene (2.1%), germacrene D (1.2%), elemol acetate (1.2%), and β-cudesmol (1.1%) were also identified.GC-MS analysis of AEO showed identification of 16 mono and sesquiterpenes, accounting for 98.2% of the total oil. Oxygenated terpenes were the major constituents (94.1%) of the oil with the β-asarone being the highest contributor (85.4 ± 1.1%). Methyl isoeugenol (3.1 ± 0.2%), caryophyllene (2.4 ± 0.3%), α-asarone (1.9 ± 0.3%), spathulenol (1.7 ± 0.1%), caryophyllene oxide (1.4 ± 0.1%), and o-cymene (5.1 ± 0.2%), camphor (3.8 ± 0.5%), and α-pinene (2.7 ± 0.2%) were other important terpenes identified in WEO. Sesquiterpenoids detected in WEO were β-caryophyllene (8.2 ± 1.0%), farnesol (3.0 ± 0.2%), and δ-elemene (2.0 ± 0.3%). Volatile composition of PEO showed various peaks in TIC of GC-MS, representing 15 constituents contributing 97.1% of the oil. Sesquiterpene constituents were highly abundant. Among these, α-guaiene (40.6 ± 1.3%) was the major compound followed by α-bulnesene (14.6 ± 0.9%), α-patchoulene (10.7 ± 0.5%), patchoulol (6.7 ± 0.3%), and β-patchoulene (5.8 ± 0.5%). A comprehensive profile of the chemical composition of the EOs, number of identified compounds, and their group-wise classification is presented in Total ion chromatogram (TIC) of WEO in GC-MS analysis exhibited characteristic peaks corresponding to 29 mono- and sesquiterpenes, contributing 97.7% of the oil. Oxygenated terpenoids (69.7%) formed the major share of the composition; borneol acetate (26.6 ± 1.7%) and β-terpineol (16.2 ± 0.9%) being the most dominant ones. 1,8-Cineol (10.6 ± 0.5%), linalool (6.4 ± 0.4%), sabinene (5.6 ± 0.3%), M. incognita at different test concentrations. Antinemic activity of the EOs is depicted in 50 of 325.41, 87.27, and 43.22 μg ml–1 concentration after 24, 48, and 72-h exposure, respectively. However, CEO containing a high amount of citronellal showed moderate activity with an LC50 of 124.50 μg ml–1 after 72 h. OEO rich in l-limonene was found to exhibit a comparatively higher nematode toxicity with a lethal concentration LC50 of 353.20 μg ml–1 within 24 h of J2 exposure. The nematicidal activity of OEO enhanced with the exposure time, and the highest activity was recorded at an LC50 of 79.35 and 39.37 μg ml–1 after 48 and 72 h, respectively  below 50 μg ml–1 except TEO, were subjected to molecular docking analysis, to understand their possible interaction with proteins for nematicidal action.In this study, TEO and AEO were found effective with an LCM. incognita were screened against the biomolecules of OEO, CNEO, and TEO, the three most effective EOs in the present study. Gibb’s free energy of binding and other docking parameters of the screened targets are presented in M. incognita J2s was best explained by the in silico inhibition of the odorant response gene 1 (ODR1). The binding pocket of the ODR1 allosteric site is composed of 45 amino acid residues. Screening of the compounds present in the three EOs against the ODR1 gave significantly low binding free energy values ranging from -36.9 to 15.3 kcal mol–1, suggestive of formation of stable protein–ligand complexes.Seven receptor proteins (putative target proteins) of –1), as depicted in The relative stability of the docked complexes of 49 ligands  with the ODR1 was computed in terms of ligand efficiency . It can –1. In this case, the amino acid residues responsible for the ligand binding interactions belonged to both guanylate cyclase and tyrosine/serine/threonine kinase catalytic domains.The citronellal–ODR1 complex with two H bonds  and five hydrophobic bonds  , exhibit–1 binding energy. One conventional H bond and three hydrophobic bonds  attributed to complex formation  in OEO showed significant inhibition of the ODR1 gene . The l-limonene–ODR1 complex exhibited seven hydrophobic interactions  in between the protein and the ligand . The γ-terpinene–ODR1 complex showed 10 hydrophobic interactions . The γ-terpinene molecule bound to the HNOBA and guanylate cyclase catalytic domains.The next major EO constituent showing significantly low free energy of binding was γ-terpinene . Inspite of this, the highest observed in vitro nematicidal activity of the OEO oil with l-limonene could be possible due to the exceptionally high content of l-limonene (93.7%). The major constituents in other active EOs was up to about 36% only  calculated for the 49 phytochemical constituents of the three oils varied in the range of 0.8–0.24 kcal molM. incognita. The previously investigated reports on OEO suggested the most prominent monoterpene, l-limonene, with a range of 32–98%  and citronella (CNEO) oils were identified in the present work as most effective for immobilization and killing of nematodes. In silico analysis suggested a higher binding capacity of geraniol, β-terpineol, citronellal, l-limonene, γ-terpinene, to the selected target proteins. Molecular docking-based understanding of the bioactivity of aromatic oils is a novel attempt toward logic-driven selection of natural materials and discovery of biopesticidal leads. The present findings will be further confirmed through wet lab molecular studies and utilized in bionematicide product development.The present study employs analytical and molecular modeling tools to relate the nematicidal activity of potential essential oils and the interactions of their chemical constituents with the target site proteins of the organism. Among the nine essential oils screened against The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.AS, AdK, AD, and AM conceptualized the study. AdK, AM, AnK, and SD validated the study. AdK, AD, AM, LN, RP, MM, JA, NP, and PS conducted the investigation. AS provided the resources. AdK, AM, AD, and SM wrote and prepared the original draft. AdK, AM, AD, SS, and RK wrote, reviewed, and edited the manuscript. AS and UR supervised the study. AS acquired the funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dexmedetomidine (Dex) was reported to reduce oxidative stress and protect against myocardial Ischemia/Reperfusion (I/R) injury. However, the molecular mechanism involved in its antioxidant property is not fully elucidated. The present study was aimed at investigating whether the Trx1/Akt pathway participated in the cardioprotective effect of Dex. In the present study, I/R-induced myocardial injury in isolated rat hearts and OGD/R-induced injury in H9c2 cardiomyocytes were established. Our findings suggested that Dex ameliorated myocardial I/R injury by improving cardiac function, reducing myocardial apoptosis and oxidative stress, which was manifested by increased GSH and SOD contents, decreased ROS level, and MDA generation in both the isolated rat hearts and OGD/R-treated H9C2 cells. More importantly, it was found that the level of Trx1 was preserved, and Akt phosphorylation was significantly upregulated by Dex treatment. However, these effects of Dex were abolished by PX-12 (a specific Trx1 inhibitor) administration. Taken together, this study suggests that Dex plays a protective role in myocardial I/R injury, improves cardiac function, and relieves oxidative stress and cell apoptosis. Furthermore, our results present a novel signaling mechanism that the cardioprotective effect of Dex is at least partly achieved through the Trx1-dependent Akt pathway. Ischemic heart disease is the leading cause of death in the whole world . AlthougDexmedetomidine (Dex), a highly selective alpha 2 adrenergic receptor agonist, is widely used for ICU sedation and auxiliary anesthesia. Interestingly, it has been found that this sedative exerts a capability of protection directly against I/R injury in multiple organs. Recent studies showed that Dex could inhibit the release of proinflammatory cytokines and attenuate I/R-related apoptosis in acute kidney injury, cerebral ischemia injury, and hepatic I/R injury, which might be ascribed to reducing the production of ROS –7.The myocardium has endogenous reducing mechanisms that are involved in the cell's counteraction against this oxidative damage. Of them, thioredoxin 1 (Trx1) is a critical molecule to protect the heart against I/R injury and ROS damage , 9. UsinBased on the above observations, we hypothesized that Dex could reduce oxidative stress and cell apoptosis by regulation of the signaling pathway Trx1/Akt, thus protecting the heart against I/R injury. In the present study, we investigated the effect of Dex pretreatment on oxidative stress and cell apoptosis in myocardial I/R models. Furthermore, the underlying mechanism of the Trx1/Akt pathway involved in Dex against I/R injury was explored.Adult male Sprague-Dawley rats (250–300 g) were purchased from the Hunan Weasley Scene of Experimental Animals Co., Ltd. (Hunan China). All animal protocols were performed following the U.S. National Institutes of Health guidelines  and approved by the Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology. Dex was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd. , and 1-methyl propyl 2-imidazolyl disulfide (PX-12) was purchased from Cayman Chemical .n = 8): control group—the hearts were perfused continuously for 120 min; I/R (I/R) group—the hearts were perfused for a 30 min stabilization and then underwent 30 min ischemia, followed by 60 min reperfusion; and I/R+Dex group—the hearts were perfused with 10 nM Dex during 30 min stabilization, and then, the rest of the procedures were the same as in the I/R group. During perfusion, hemodynamic parameters were recorded continuously via a water-filled balloon placed into the left ventricle and attached to a pressure transducer after the heart was mounted on a Langendorff apparatus . The data were collected and analyzed using a PowerLab data acquisition system with LabChart 7 software . Measured parameters included left ventricle developed pressure (LVDP), left ventricle end-diastolic pressure (LVEDP), and maximum LVDP increase (+dp/dt) and decrease (−dp/dt) rates. At the end of the perfusion, hearts were collected for the determination of the following experiments.A myocardial I/R model was established as previously described . Briefly2, 95% air, 37°C). To mimic the I/R injury model on H9C2 cells, the oxygen-glucose deprivation/reperfusion (OGD/R) model was generated by bathing the cells in glucose-free DMEM within an anaerobic chamber  at 37°C for 12 h (oxygen-glucose deprivation (OGD)). Then, the cells were incubated with normal culture medium under normoxic conditions  for 6 h at 37°C (reperfusion (R)). Normoxic control cells (control group) were treated identically without OGD/R. Furthermore, H9C2 cells were exposed to OGD/R with or without Dex preconditioning (1 μM) for 1 h. The Trx1 inhibitor PX-12 (5 μM) was pretreated for 30 min before OGD/R.The H9C2 cardiac cell line was obtained from ATCC and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified incubator  for 15 min at 37°C. The sections were fixed in 4% paraformaldehyde-phosphate-buffered saline (PBS) solution at 4°C. Normal tissue was stained red while the infarcted tissue was white (unstained). Infarct size was scored using the ImageJ software  and was expressed as a percentage of the total ventricular area.Apoptosis was evaluated using the terminal dUTP nick-end labeling (TUNEL) staining kit  based on the manufacturer's instructions. Images were captured using a light microscope , and the apoptotic index was represented as the ratio of the number of apoptotic nuclei to the total number of nuclei counted ×100%.The hearts were collected and homogenized in RIPA lysis buffer. After centrifugation at 12,000 g for 10 min, the supernatant was collected. Then, contents of glutathione (GSH) and malondialdehyde (MDA), as well as activity of the antioxidative enzyme superoxide dismutase (SOD), were measured according to the instructions of the corresponding assay kit . Besides, the process of analysis in H9c2 cells was similar to the method described above.μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in the dark for 20 min at 37°C, and washed three times with serum-free medium. DCFH-DA fluorescence was detected at 525 nm using flow cytometry.Cellular ROS production was assayed using the ROS assay kit . Briefly, H9C2 cells were treated according to the conditions indicated, incubated with 10 In an in vitro study, cell viability was evaluated using the 3--2,5-diphenyltetrazolium bromide (MTT) assay kit  according to the manufacturer's instructions. Optical density (OD) was measured using a microplate reader  at 568 nm.P < 0.05 was considered statistically significant.All data were presented as the mean ± standard deviation (SD) value of at least three independent experiments. Statistical analyses were conducted using GraphPad Prism 7 . Data were analyzed by a one-way ANOVA (two-way ANOVA for the isolated heart perfusion study) followed by post hoc Tukey's test for multiple comparisons. P < 0.01, respectively; LVEDP P < 0.05). Meanwhile, Dex dramatically improved the recovery of LVDP (54.71 ± 3.93 mmHg), +dp/dtmax (1722 ± 26.23 mmHg/s), −dp/dtmin (1082 ± 68.57 mmHg/s), and LVEDP (25.24 ± 2.54 mmHg) compared with I/R alone . At the end of reperfusion, there were no differences between groups Dex and control in these parameters.The influence of Dex treatment on the recovery of cardiac function following I/R was investigated. As shown in P < 0.01, respectively, Figures P < 0.01, P < 0.01, To examine the effects of Dex on myocardial I/R injury in rats, myocardial tissue injury was detected by TTC staining and TUNEL assay. The results manifested that I/R injury significantly increased the apoptosis rate in myocardial cells from 4.36 ± 2.32% (control) to 28.24 ± 4.31% (I/R). Moreover, the infarct size percentage was increased to 30.37 ± 4.83% compared with 5.05 ± 2.06% in nonischemic controls . In contrast, MDA was elevated markedly after I/R . However, Dex treatment enhanced GSH level (41.17 ± 7.11 μmol/g) and SOD activity (12.54 ± 2.87 U/mg)  and significantly decreased MDA level (4.28 ± 0.79 μmol/mg) in the ischemic myocardium .To investigate the effect of Dex treatment on I/R-induced oxidative stress, the changes of three critical indicators of oxidative stress were measured, including the content of MDA, GSH, and activity of SOD. As shown in P < 0.01, P < 0.01, P < 0.05, P < 0.01, To further clarify the underlying mechanism, we investigated the role of Dex in Trx1 and Akt phosphorylation in myocardial I/R. The result of Western blotting showed that after I/R injury, Trx1 expression decreased significantly by 0.54-fold compared with the control  in our vitro experiment using H9c2 cardiomyocytes treated with OGD/R. OGD/R induced 27.33 ± 4.74% of cell death compared to normoxic conditions (3.37 ± 2.40%) and reduced cell viability to 59.22 ± 3.55% (μmol/mg), decreased GSH level (29.81 ± 3.47 μmol/g), and SOD activity (16.93 ± 1.70 U/mg) . Dex significantly reduced the ROS level by 1.52-fold and decreased MDA content (20.06 ± 2.15 μmol/mg), as compared with the OGD/R group  and SOD activity (25.97 ± 3.46 U/mg) , with enhanced MDA formation (28.41 ± 4.12 μmol/mg), reduced level of GSH (23.36 ± 3.62 μmol/g), and SOD activity (16.36 ± 2.92 U/mg) . Nevertheless, these effects were reversed by PX-12 . The application of dexmedetomidine in cardiac surgery could reduce patients' risks of abnormal hemodynamics. It also benefited patients with cardiac disease undergoing noncardiac surgeries and resulted in less postoperation complications [Dex is a highly selective ications , 26. Theications , 15, 16.While we have proven Dex's myocardial protective effect and identified the Trx1/Akt signaling pathway's possible involvement in this study, several major limitations should be noted. First, this study's findings were obtained from a cellular and an isolated heart I/R model, which are needed to be proven in further in vivo experiments. Second, although we have found that Dex could preserve the level of Trx1 and enhance the activation of Akt in myocardial I/R injury, the underlying interaction mechanism between Dex and Trx1 is still unknown and should be further investigated.α2 receptor agonist, protects against myocardial I/R. Specifically, it maintains the capability to improve cardiac function in I/R injury, relieve oxidative stress, and reduce cell apoptosis. Furthermore, the protective effects of DEX in I/R injury are achieved partly through the Trx1-dependent Akt pathway.In conclusion, our work revealed that DEX, an


Outcome in high-risk patients with refractory or relapsed germ cell tumours (GCT) remains poor. Novel strategies enhancing therapeutic efficacy whilst limiting therapeutic burden are warranted, yet immunotherapy approaches geared towards activating endogenous antitumor responses have not been successful thus far. Redirection of cytotoxic effector cells by bispecific antibodies represents a promising approach in this setting. We demonstrate that the Epithelial Cell Adhesion Molecule (EpCAM) is broadly expressed in GCT cell lines of different histologic origin including seminoma, choriocarcinoma (CHC), and embryonal carcinoma (EC). In these GCT lines of variable EpCAM surface expression, targeting T cells by the prototypic bispecific EpCAM/CD3-antibody (bAb) Catumaxomab together with natural killer (NK) cell engagement via the Fc domain promotes profound cytotoxicity across a broad range of antibody dilutions. In contrast, tumor cell lysis mediated by either immune cell subset alone is influenced by surface density of the target antigen. In the CHC line JAR, NK cell-dependent cytotoxicity dominates, which may be attributed to differential surface expression of immunomodulatory proteins such as MHC-I, CD24, and Fas receptors on CHC and EC. In view of redirecting T cell therapy mediated by bispecific antibodies, such differences in GCT immunophenotype potentially favoring immune escape are worth further investigation. Malignant Germ cell tumors (GCT) constitute a heterogeneous group of tumors comprising undifferentiated seminoma as well as embryonal carcinoma (EC), choriocarcinoma (CHC), yolk sac tumor (YST), or teratoma components . GCT areIn cancer therapy, improved understanding of interactions between tumor and immune cells has paved the way for establishing effective immunotherapeutic approaches. While in the past GCT have long been considered to mimic testicular immune privilege, consolidating evidence has revealed infiltration of activated cytotoxic and memory T cells as well as macrophages predominately in seminoma with helper T cells and natural killer (NK) cells being rarely present ,11,12,13Amongst bispecific antibodies (bAb), constructs that comprise dual binding sites for both CD3 and a suitable target antigen on tumor cells are attracting increasing attention as strategies of antineoplastic immunotherapy. They redirect T cells to tumor cells promoting cytotoxic synapse formation and subsequent tumor cell lysis. In addition, via their Fc-domain, bAb enlist accessory cells such as natural killer (NK) cells and macrophages into the immune response, complementing T cell-dependent tumor destruction .In GCT, the transmembrane epithelial adhesion molecule  constitutes a promising target for such an approach as it is broadly present on the surface of GCT of different origins irrespective of age, sex, site, and clinical tumor stage . EpCAM eHere, we demonstrate that, in GCT cell lines of different histology, the EpCAM-directed prototypic bAb Catumaxomab facilitates recruitment and activation of accessory cells in addition to redirected T cells and promotes highly efficacious antineoplastic toxicity. We also delineate characteristics in GCT phenotype potentially favoring immune escape that are worth further investigation.Levels of EpCAM protein on the surface of different GCT cell lines were assessed by flow cytometry a. EpCAM EpCAM expression analysis  and fibroblasts (MPAF). CD133, which combined with EpCAM can be indicative for cancer stem cells, is expressed to high levels on the seminoma cell line TCam-2 and the EC lines GCT27 and NCCIT. CD133 is detected only on half of the cells in the nullipotent EC line 2102Ep and is absent on the CHC line JAR  including T, NK, and B cells as well as monocytes and either the bispecific trifunctional EpCAM antibody Catumaxomab (bAb) or the monoclonal EpCAM antibody Vu1D9 mAb; a,b.p < 0.001) and decreases with each dilution step. Thus, cell death is 18.4 ± 7.4% at 1 µg/mL and only 3.1 ± 2.1% at 0.01 µg/mL mAb.PBMC alone had no cytotoxic effect on 2102Ep cells. In contrast, at an effector-to-target (E:T) ratio of 25:1, bAb-induced tumor cell lysis is 44.9 ± 2.5% at 1 µg/mL and 44.2 ± 5.4% at 0.01 µg/mL bAb. Even with further reduction of bAb concentration down to 0.0001 µg/mL, tumor cell lysis is still 35.8 ± 6.9% a. In thep < 0.001). Furthermore, cytolytic activity of the bAb persists at high levels across the entire drug concentration range, with 55.1% ± 5.7% at 1 µg/mL bAb and with 57.7 ± 6.0% and 53.6 ± 7.4% when treated with 0.01 µg/mL and 0.0001 µg/mL bAb, respectively. Upon incubation with the mAb in the presence of PBMC, only 34.7 ± 10.6% of 2102Ep cells die at 1 µg/mL and 10.7 ± 2.2% die at 0.01 µg/mL.Increasing the E:T ratio to 50:1 enhances both bAb- and mAb-mediated cellular kill b. Again,p < 0.001) and successively declines with each dilution step from 63.0 ± 3.4% at 1 µg/mL to 33.9 ± 6.4% at 0.01 µg/mL and only 4.0 ± 3.3% at 0.0001 µg/mL.Prolongation of the incubation period further improves the cytotoxic effect of both the bAb and mAb c. Again,p < 0.005) with only 11.4 ± 3.6% at 1 µg/mL, 9.9 ± 2.2% at 0.1 µg/mL, and 11.6 ± 4.1% at 0.001 µg/mL mAb .Even in the pluripotent EC line NCCIT b displayp = 0.23; 0.1 µg/mL: p = 0.18; 0.001 µg/mL: p = 0.15).Of note, in the CHC cell line JAR, bAb-induced cellular kill does not exceed mAb-mediated cytotoxicity c. In spiTo allocate antibody-mediated cytotoxicity to different effector cell subsets, NK and T cells were isolated from buffy coats and incubated separately at an E:T ratio of 20:1 either with the highly EpCAM-positive EC line 2102Ep or the EC line NCCIT expressing EpCAM only on 50% of tumor cells. Cytotoxicity was assessed by europium release assay after 4 h of incubation with the bispecific and monospecific antibodies a,b.p < 0.001). Thus, cell death in 2102Ep reaches 91.8 ± 0.7% at 1 µg/mL bAb and 90.5 ± 3.5% at 0.1 µg/mL bAb. Further reduction of the bAb concentration results in a drop of NK cell-mediated cell death down to 21.1 ± 5.9% at 0.001 µg/mL bAb, which is no longer statistically different to NK cell-mediated cell death in the presence of mAb at this concentration (p = 0.43).In the absence of a specific binding site for T cells, EpCAM-mAb facilitates only NK but no T-cell dependent cytotoxicity. In the EpCAM-“high” EC line 2102EP, mAb-mediated cell death is substantial at the highest mAb concentration in the presence of NK cells but declines from 44.6 ± 2.7% at 1 µg/mL mAb to 28.9 ± 4.1% at 0.1 µg/mL and 18.2 ± 3.5% at 0.001 µg/mL mAb a. The bip < 0.001).In contrast, following incubation with isolated T cells a, inductp < 0.001 each). At 0.001 µg/mL bAb, cellular lysis is negligible with 2.7 ± 0.8% without statistical difference to the mAb at the same drug concentration (p = 0.9). In the EpCAM-“low” EC line NCCIT, T cell-dependent cytotoxicity in the presence of bAb significantly exceeds NK cell-mediated cell death (p < 0.001). Cellular kill declines in a dose-dependent manner from 57.6 ± 5.3% at 1 µg/mL bAb to 44.7 ± 2.3% at 0.01 µg/mL and 20.8 ± 1.4% at 0.001 µg/mL bAb.In NCCIT with only 50% of EpCAM-positive cells, the EpCAM-binding mAb fails to mediate relevant NK cell-mediated cytotoxicity. Even at the highest concentration of 1 µg/mL mAb, only 3.4 ± 3.0% of tumor cells are killed after incubation with NK cells b. In conll death b at eachExpression of CD44 mediating T cell migration and adhesion is high in all GCT lines analyzed, while the costimulatory molecule CD24 is expressed to high levels on EC but is absent in the CHC line JAR . MHC-I eIn GCT, diverse immune cell infiltration of the tumor microenvironment reflects the heterogeneous histology of this group of neoplasms, ranging from virtual absence of immune cells in CHC to intermediate T cell accumulation in EC and YST and to dense inflammatory immune cell infiltration in gonadal dysgerminoma and seminoma. Thus, immunotherapeutic agents such as bispecific antibodies that specifically target malignant cells and redirect T cells to the tumor microenvironment constitute a promising approach to harness the immune system for antineoplastic protection in GCT.EpCAM mRNA and EpCAM protein levels in GCT of different histologies [Here, we extend our previous findings of tologies  and demotologies . Catumaxtologies ,24. Of ntologies . Converstologies . Here, wIt is noteworthy that, in GCT of different histologies, cytotoxicity facilitated by the EpCAM/CD3-binding bAb Catumaxomab is stable over a broad range of antibody concentrations from 1 µg/mL down to 0.0001 µg/mL, the latter corresponding to plasma levels achieved after intravenous administration of the lowest dose in a phase I dose-escalation study . In vitrIn contrast, in the CHC line JAR, the bAb fails to trigger tumor cell lysis by isolated T cells alone whilst equivalent cytotoxic efficacy of the bispecific and monoclonal antibody indicates predominantly NK cell-mediated tumor cell destruction. As a consequence, the overall sensitivity of the CHC cell line JAR to antibody-mediated lysis is limited. MHC subclasses can function as killer cell inhibitory receptors, and their absence as a phenotypic characteristic in CHC might contribute to promoting NK cell activity whilst impeding T cell responses. In addition, lack of MHC-I as well as costimulatory molecules is one of the key mechanisms by which tumor cells escape antigen-specific immune surveillance. In contrast to EC lines, JAR lacks CD24 expression, a key factor of T cell homeostasis and proliferation . ChoriocIn many cancers, PD-1/PD-L1 expression on neoplastic and other cellular components of the tumor microenvironment has been recognized as key to tumor cell-mediated suppression of the antineoplastic immune response. Immunohistochemical analysis of testicular GCT revealed variable expression of PD-L1 and CTLA-4, with PD-L1 expression in GCT being significantly higher compared to normal testicular tissue . In addiConcerning bAb-treatment, this is of interest as, similar to attenuation of MHC-dependent cytotoxic T cell responses, T cells redirected to EpCAM-positive target cells may become subdued by various mechanisms of immune inhibition such as PD-1/PD-L1 and CTLA-4/CD80/86 . Indeed,GCT, despite their characteristically low mutational burden and lack of neoantigens, express proteins characteristic of their embryonal origin such as Alpha-fetoprotein (AFP) with documented MHC-I-restricted, AFP-directed antineoplastic T cell responses in other AFP-positive cancers ,39. The Broad activation of cellular immunological effectors however carries the risk of nonspecific off-target effects. Thus, following treatment with the bAb Catumaxomab, it is the nonspecific binding of the Fc-domain to FcγR-positive Kupffer cells that was found to be the cause for the observed antibody-mediated hepatoxicity . BispeciLikewise, in T cells retrovirally modified to express target antigen-specific chimeric T-cell receptors (CAR), incorporation of suicide genes such as inducible caspase-9 in vector constructs allows for optional induction of T cell apoptosis to this end . Indeed,Currently, there is a surge of development of bAb and CAR-T cells with over two hundred constructs in early clinical testing. Among these are two EpCAM-recognizing bispecific antibodies and two CAR-T cell constructs . In viewJAR (HTB-144), GCT27, and NCCIT were purchased from American Type Culture Collection . TCam-2 and 2102EP cells were kindly provided by L.Looijenga (Princess Máxima Center for Pediatric Oncology/NL). Cell lines were cultivated as described previously ,48.TM flow cytometer , and analyzed with Cytometry List Mode Data Acquisition and Analysis Software (Beckman Coulter Inc.).Monoclonal antihuman-EpCAM , -CD133 , -CD44 , -CD24 , -HLA-ABC , -CD95 , -FasL , and -PD-L1  as well as mouse-IgG1 antibodies as isotype controls  were used for flow cytometry. Collected cells were first washed with PBS + 2% FCS (FACS buffer), stained with the fluorochrome-conjugated monoclonal antibodies, and incubated for 15 min at room temperature. Upon washing to remove unbound reagents, cells were resuspended in FACS buffer, acquired on a NaviosEpCAM expression: forward 5′-GCAGCTCAGGAAGAATGTG-3′, reverse 5′-CAGCCAGCTTTGAGCAAATGAC-3′. GAPDH was used as a housekeeper and for data normalization with the following GAPDH primers being employed: forward 5′-TGCCAAATATGATGACATCAAGAA-3′, reverse 5′-GGAGTGGGTGTCGCTGTTG-3′. A melting point analysis was performed to confirm primer specificity. PCR was performed at 94 °C/30 s and 60 °C/60 s for 40 cycles using the ViiA 7 Real-Time PCR System .Quantitative real-time RT-PCT (qRT-PCR) was performed as described previously . The folTM, Cedarlane, Burlington, Canada). NK cells were negatively selected from PBMCs using the untouched NK cell isolation kit (human) onto LD columns , according to the manufacturer’s instructions. Purity of enriched NK cells was assessed by flow cytometry. For isolation of T cells, 10 mL of Buffy coat was incubated with 500 µL RosetteSepTM Human T Cell Enrichment Cocktail  and proceeded according to the manufacturer’s instructions. Blood samples from healthy donors were collected by the local institute of Experimental Haematology and Transfusion medicine (IHT) at the University Hospital of Bonn. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll–Hypaque density gradient centrifugation  in 1 liter of distilled water and was adjusted to pH 7.4. Upon sterile filtration, 100 mM Europium(III)chloride dissolved in distillated water was added to the labelling buffer. Tumor cells (5 × 106) from the GCT cell lines 2102Ep, GCT27, NCCIT, or JAR were incubated 10 min. on ice with Europium-labelling buffer. Then, target cells were permeabilized by electroporation using the Lonza NucleofectorTM II with SCN Basic Neuro Program 8  and washed four times with RPMI 1640 20% FCS. All chemicals for the Europium labeling buffer were of analytical grade provided by Sigma-Aldrich (Germany). Labeling buffer contained 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 93 mM NaCl, 5 mM KCl, 2 mM MgCl3 cells/100 µL medium and dispensed into wells of 96-well roundbottomed microtiter plates. An equal volume of effector cells was added to each well. Suspensions of effector cells were adjusted to give effector/target (E/T) ratios ranging of 50:1 and 25:1 for PBMC and 20:1 for isolated NK and T cells, respectively. Monoclonal antihuman EpCAM-antibody  or trifunctional EpCAM/CD3-antibody Catumaxomab  were added in concentrations ranging from to 0.0001 to 1 µg/mL. Controls consisted of PBMC and media without tumor cells. All assays were performed in triplicate. The microplates were centrifuged briefly to bring effectors and targets in contact with each other and then incubated for 4 h or 8 h at 37 °C in a humidified atmosphere of 5% CO2 in air.The cytotoxicity assay is based on the Europium-DPTA-chelate (Eu-DPTA) release from the cytoplasm of lysed cells, which is quantified in the supernatant with a time-resolved fluorometer. Target cells labeled with Europium were adjusted to a concentration of 5 × 10TM, Sigma-Aldrich, St. Louis, MO, USA), and 200 μL aliquot of enhancement solution  was added to each well. After mixing for 10 min., fluorescence was measured in a time-resolved fluorometer . The percentage of specific cytotoxicity was expressed as specific release in % /(maximum release−spontaneous release) × 100). Spontaneous release was determined by incubating the targets with 100 μL of complete medium instead of effector cells, and maximum release was determined by incubating the cells with 100 μL of 0.1% Triton-X. After incubation, 20 μL aliquots of the supernatants were transferred to wells of a flat-bottom 96-well microplate . For determination of statistical significance between the cytotoxic efficacy of the bispecific compared to the monoclonal antibody, two-tailed Student´s t-test and multiple T-tests with adjusted p-values were used. p-values less than 0.05 were considered statistically significant.Calculations of mean values, standard deviation, and Although prognosis in GCT patients with cisplatin as the mainstay of treatment is excellent, there remains a subgroup of patients for whom novel therapeutic approaches are required. We demonstrate that, in GCT, the EpCAM-directed prototypic bispecific antibody Catumaxomab facilitates recruitment and activation of accessory cells in addition to redirected T cells and promotes highly efficacious antineoplastic toxicity. We also delineate differences in GCT phenotype potentially favoring immune escape that are worth further investigation. Combined effector cell redirection and blockade of inhibitory mechanisms with checkpoint inhibitors may serve to elevate attenuation of redirected antineoplastic immune responses in the future. Based on our in vitro findings in GCT and early clinical experience of EpCAM-redirected immunotherapy in other epithelial EpCAM-positive cancer entities, such therapeutics may also prove beneficial for the treatment of cisplatin-resistant GCT and warrant clinical exploration.


Toxoplasma gondii is a parasite that primarily infects through the oral route. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) play crucial roles in the immune responses generated during parasitic infection and also drive the inflammatory response against invading parasites. However, little is known about the regulation of NLRs and inflammasome activation in T. gondii-infected human small intestinal epithelial (FHs 74 Int) cells.T. gondii were subsequently evaluated for morphological changes, cytotoxicity, expression profiles of NLRs, inflammasome components, caspase-cleaved interleukins (ILs), and the mechanisms of NLRP3 and NLRP6 inflammasome activation. Immunocytochemistry, lactate dehydrogenase assay, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, and western blotting techniques were utilized for analysis.FHs 74 Int cells infected with T. gondii-infected conditions, members of the NLRs, inflammasome components and caspase-cleaved ILs were expressed in the FHs Int 74 cells, except for NLRC3, NLRP5, and NLRP9. Among the NLRs, mRNA expression of NOD2, NLRP3, NLRP6, and NAIP1 was significantly increased in T. gondii-infected cells, whereas that of NLRP2, NLRP7, and CIITA mRNAs decreased significantly in a time-dependent manner. In addition, T. gondii infection induced NLRP3, NLRP6 and NLRC4 inflammasome activation and production of IL-1β, IL-18, and IL-33 in FHs 74 Int cells. T. gondii-induced NLRP3 inflammasome activation was strongly associated with the phosphorylation of p38 MAPK; however, JNK1/2 had a weak effect. NLRP6 inflammasome activation was not related to the MAPK pathway in FHs 74 Int cells.Under normal and T. gondii-infected FHs 74 Int cells. These findings may contribute to understanding of the mucosal and innate immune responses induced by the NLRs and inflammasomes during T. gondii infection in FHs 74 Int cells.This study highlighted the expression profiles of NLRs and unraveled the underlying mechanisms of NLRP3 inflammasome activation in  Toxoplasma gondii is an obligate intracellular protozoan parasite that infects one-third of the world’s population [T. gondii or through the ingestion of materials contaminated with cat feces that may contain T. gondii oocysts. Once inside the body, the parasite breaches the intestinal epithelial barrier and spreads from the lamina propria to other organs [pulation . Infectir organs . Intestir organs . The innr organs , 5. It ir organs .T. gondii-infection mediated immune responses in the infected hosts. It has been reported that NOD2-deficient mice are unable to clear T. gondii and fail to induce an appropriate adaptive immune response [T. gondii infection [NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and NAIP are significantly elevated because of T. gondii infection, in a time-dependent manner [T. gondii infection protection [NLRs are a large group of cytosolic sensors that have diverse functions in innate immunity and inflammation. Based on the type of N-terminal domain, NLRs are classified into four subfamilies, NLRA, NLRB, NLRC, and NLRP, and an additional subfamily, NLRX1 , 8. Severesponse . In addinfection , 11. In t manner . Althougotection –12, littT. gondii infection in cells with NLRP1 knockdown fails to induce the production of inflammatory cytokines including IL-1β, IL-18, and IL-12 compared to control cells [Plasmodium sp., Trypanosoma cruzi, Leishmania sp., and T. gondii [T. gondii proliferation in small intestinal epithelial cells [T. gondii infection in various cells, information on inflammasome activation in gut epithelial cells infected with T. gondii is very scarce.Ligand recognition by the NLR family members, such as NLRP1, NLRP3, NLRP6, NLRP12, and NLRC4, leads to the activation of inflammasome, a multiprotein complex, which cleaves interleukin (IL)-1β, IL-18, IL-33, and IL-37 (IL-17A) by caspases, the effector components of inflammasomes –14. T. gol cells . The bro. gondii . The P2Xal cells . While rT. gondii-infected human small intestinal epithelial (FHs 74 Int) cells. Therefore, this study evaluated the expression profiles of NLRs, inflammasome components, and caspase-cleaved ILs and investigated the mechanisms of some popular inflammasomes’ activation in T. gondii-infected FHs 74 Int cells.NLRs play a crucial role in inducing immune responses during parasitic infection and driving the inflammatory responses against invading parasites . However2. The medium was changed every 2–3 days.A non-transformed human fetal small intestinal epithelial cell line (FHs 74 Int cells) was purchased from ATCC  and cultured in DMEM with 10% (v/v) heat-inactivated fetal bovine serum (FBS), an antibiotic–antimycotic solution, and 30 ng/ml human epidermal growth factor  at 37 °C in a humidified atmosphere at 5% (v/v) COT. gondii RFP-RH or RH strain were maintained as described previously [T. gondii at a multiplicity of infection (MOI) of 5 for 2–3 days. After spontaneous host cell rupture, parasites and cellular debris were pelleted by centrifugation and washed in cold PBS. The final pellet was resuspended and passed through a 26-gauge needle fitted with a 5.0 μm pore-sized filter .Tachyzoites of the eviously . BrieflyTotal RNA was extracted using TRIzol Reagent , and RNA was transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen) as described by the manufacturer. Polymerase chain reaction (PCR) was performed with TaKaRa Ex Taq  in reactions containing 33.75 μl distilled water, 5 μl 10 × Ex Taq buffer, 4 μl dNTP mixture (2.5 mM each), 2 μl of each primer, 0.25 μl of TaKaRa Ex Taq, and 3 μl of template cDNA to total 50 μl. PCR amplification conditions were an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, an annealing 60 °C for 30 s, and an extension step of 72 °C for 30 s. Finally, PCR was completed with the additional extension step for 10 min. The PCR products were analyzed on 1.5% agarose gel in 0.5 × TBE buffer and visualized using ethidium bromide and a UV transilluminator. The details of primers designed are presented in Table 4 cells/well and incubated for 24 h. The cells were infected with T. gondii at MOI 10 for 0, 4, and 8 h. Subsequently, the cells were washed with Hank's balanced salt solution (HBSS) and fixed with freshly prepared 4% paraformaldehyde for 1 h at room temperature. After washing five times with PBS containing 0.3% Triton X-100 (PBS-T) for 10 min, the cells were incubated with primary antibodies  for 2 h at room temperature. The cells were washed to remove excess primary antibody and then incubated with the appropriate fluorescently labeled secondary antibodies  for 2 h at room temperature. After mounting with VECTASHIELD HardSet antifade mounting medium with DAPI , fluorescence images were acquired using a confocal microscope .FHs 74 Int cells were seeded onto coverslips in 12-well plates at a density of 1 × 104 cells were seeded into 96-well plates and infected with T. gondii MOI 10 for 0, 4, and 8 h in an incubator . Next, 50 µl of the supernatant was transferred into a new 96-well plate, and 50 µl of CytoTox 96 reagent was added and incubated for 30 min at room temperature. After incubation, the absorbance of the solution was measured immediately at 490 nm using a microplate reader . LDH levels in the media were quantified and compared to control values according to the kit instructions.LDH assay was performed to quantify cytotoxicity. This assay was conducted using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's protocol. Briefly, 1 × 10® Green PCR Master Mix . The primers used in this study are summarized in Table –ΔΔCt method.qRT-PCR was performed using Power SYBRT. gondii at MOI 10 for 0, 4, and 8 h. FHs 74 Int cells were preincubated with the 30 μM of SB203580 (p38 MAPK inhibitor) and SP600125 (JNK1/2 inhibitor) for 2 h and infected with T. gondii MOI 10 for a further 8 h. Subsequently, cell lysates were collected and lysed in ice-cold radio-immunoprecipitation assay (RIPA) buffer . Protein concentrations were determined using the Bradford assay . Total protein (30 μg) was resolved on 10–12% SDS-PAGE gels and then transferred to PVDF membranes . The membranes were blocked with 5% nonfat skim milk in TBS containing 0.1% Tween 20 (TBST) for 1 h and incubated with primary antibodies against NLRP1, NLRP3, NLRP6, NLRC4, NAIP1, cleaved caspase-1, ASC, cleaved IL-1β, IL-33, p-p38 MAPK, p38 MAPK, p-ERK1/2, ERK1/2, p-JNK1/2, JNK1/2, TP3, and α-tubulin overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology) for 2 h at room temperature. The membrane was soaked with Immobilon Western Chemiluminescent HRP Substrate (Jackson ImmunoResearch Laboratories), and chemiluminescence was detected with a Fusion Solo System . Band intensity was quantified using ImageJ software . The result was normalized to the α-tubulin protein level and expressed as fold changes compared to the control group.FHs 74 Int cells were infected with T. gondii at MOI 10 for 0, 4, and 8 h. The supernatants from the mock- or T. gondii-infected FHs 74 Int cells were collected in triplicate, and IL-1β and IL-18 levels were measured using commercially available ELISA kits following the manufacturer’s instructions . The cytokine concentrations in the samples were calculated from standard curves obtained using recombinant cytokines.FHs 74 Int cells were infected with P < 0.05.All results are presented as the means ± standard deviations (SDs) of at least three independent experiments, unless otherwise indicated. Statistical comparisons were carried out using GraphPad Prism software , and multiple t-tests was used to determine one-way ANOVA procedures. Differences were considered significant at NLRC3, NLRP5, and NLRP9 mRNAs .FHs 74 Int cells were incubated with T. gondii-induced cytotoxicity of FHs 74 Int cells, the cells were incubated with T. gondii at an MOI of 10 for 0, 4, and 8 h. Post-incubation, LDH assay was performed. Release of LDH significantly increased in the T. gondii-infected groups compared to that in the mock-infected control group. Cytotoxicities of FHs 74 Int cells infected with T. gondii for 0, 4, and 8 h were 4.34 ± 0.15%, 19.21 ± 1.88%, and 40.02 ± 1.57%, respectively . Neither normal nor T. gondii-infected FHs 74 Int cells expressed NLRP5 mRNA. While T. gondii infection increased the expression of mRNAs encoding caspase-1, ASC, IL-1β, IL-18, and IL-33, it had no effect on the expression of mRNAs encoding caspase-5, caspase-8, and IL-37  and SP600125 (JNK inhibitor) significantly decreased the phosphorylation of p38 MAPK and JNK1/2 in T. gondii-infected cells compared with that in inhibitor-untreated T. gondii-infected cells. Interestingly, pretreatment with SB203580 significantly downregulated T. gondii infection-induced NLRP3 expression; however, the effects of SP600125 pretreatment were considerably less than those of SB203580 pretreatment. Pretreatment with SB203580 and SP600125 had no effect on the regulation of T. gondii-induced NLRP6 activation. SB203580 and SP600125 pretreatment significantly attenuated T. gondii-induced elevation in the cleaved IL-1β and cleaved IL-18 levels. Furthermore, pretreatment with SB203580 and SP600125 considerably upregulated the T. gondii antibody (TP3) level in T. gondii-infected FHs 74 Int cells compared with that in untreated T. gondii-infected cells  pathway is associated with inflammasome activation , 19. HenT. gondii-induced NLRP3 inflammasome production in T. gondii-infected small intestinal epithelial FHs 74 Int cells, although the p38 MAPK and JNK1/2 pathways are involved in NLRP3 activation. However, NLRP6 inflammasome activation was not related to the MAPK pathway in FHs 74 Int cells , and activation of apoptosis through interaction with host inflammasomes [Neospora caninum-induced NADPH-dependent ROS generation plays an important role in NLRP3 inflammasome activation [Expression of the NLR family as a pattern recognition receptor is cell specific; however, little was known about the regulation of NLRs and their activation mechanisms in intestinal epithelial cells. In this study, the expression patterns of the NLRs, inflammasome components, and caspase-cleaved ILs were varied in the FHs Int 74 cells under normal and al cells , this is stimuli , 7. Thusmmasomes . In additivation .T. gondii-infected FHs 74 Int cells revealed that T. gondii infection induces the expression of NLRP3, ASC, NLRP6, NLRC4, cleaved caspase-1, and cleaved caspase-8. In addition, increased production of ILs such as IL-1β, IL-18, and IL-33 was observed in T. gondii-infected FHs 74 Int cells. These findings suggest that T. gondii infection induces the activation of NLRP3, NLRP6, and NRC4 inflammasomes via the recruitment of ASC and caspases and production of proinflammatory cytokines in FHs 74 Int cells. These findings are partially consistent with those of a previous study, that is, NLRP3, ASC, caspase-1, and IL-1β were detected in T. gondii-infected mice [T. gondii infection of THP-1 macrophages increased the production of IL-1β and inflammasome sensors, including NLRP1, NLRP3, NLRC4, NLRP6, NLRP8, NLRP13, AIM2, and NAIP, in a time-dependent manner [The present investigation in ted mice . The fint manner . CompareT. gondii-infected FHs 74 Int cells by pretreatment with SB203580 and SP600125, inhibitors of p38 MAPK and JNK, respectively. While SB203580 pretreatment significantly downregulated T. gondii-induced expression of NLRP3, cleaved IL-1β, and cleaved IL-18, the effects of SP600125 pretreatment were less than those of SB203580 pretreatment. However, NLRP6 activation was not affected by pretreatment with SB203580 or SP600125 in T. gondii-infected FHs 74 Int cells. These findings suggest that p38 MAPK signaling is a more important factor than JNK1/2 signaling, although both signaling pathways were involved with NLPR3 activation in T. gondii-infected FHs 74 Int cells. However, NLRP6 inflammasome activation had no correlation with the MAPK pathway.MAPKs, highly conserved in all eukaryotes, control a variety of cellular processes, including cell differentiation, proliferation, survival, and stress responses. It has been reported that the MAPK pathway is associated with inflammasome activation , 20, 22.T. gondii-infected FHs 74 Int cells after pretreatment with SB203580 or SP600125 to evaluate the host cell environment for parasite proliferation. Both inhibitors apparently increased the parasite proliferation compared with that in the untreated group of T. gondii-infected FHs 74 Int cells. However, SB203580 pretreatment induced higher TP3 expression than SP600125 pretreatment in T. gondii-infected FHs 74 Int cells, and this was opposite to the expression pattern of NLRP3 inflammasome and IL-1β and IL-18 proteins after pretreatment with SB203580 and SP600125. These data suggest that NLRP3 inflammasome is important for restricting T. gondii proliferation in T. gondii-infected FHs 74 Int cells, and the p38 MAPK pathway is a more critical factor than the JNK1/2 pathway in the regulation of T. gondii-induced NLRP3 inflammasome production. Our results are consistent with those of previous studies, that is, T. gondii triggers NLRP3 or NLRP1, which leads to parasite restriction and resistance to toxoplasmosis [T. gondii infection significantly inhibited T. gondii tachyzoite replication in fibroblasts. These different effects on T. gondii growth may be a result of differences in cell type, treatment method, time of inhibitor treatment, T. gondii infection time, and host cell microenvironments.We also compared the parasite levels in lasmosis , 16, andlasmosis . Howeverlasmosis , who repT. gondii-infected human small intestinal epithelial (FHs 74 Int) cells. T. gondii infection induced the expression of NLRs, inflammasome components, and caspase-cleaved ILs in the FHs Int 74 cells, but their expression patterns were varied. NLRP3 inflammasome activation was strongly associated with the p38 MAPK pathway; however, NLRP6 inflammasome activation had no correlation with the MAPK pathway. We believe that the study findings will contribute to the understanding of mucosal and innate immune responses induced by NLRs and inflammasomes in T. gondii-infected FHs Int 74 cells.We demonstrated the regulation of NLRs and NLR-related inflammasome activation in


Higher dietary intake of calcium (Ca), magnesium (Mg), and vitamin D has been associated with reduced risk of type 2 diabetes (T2DM), and a higher intracellular ratio of Ca to Mg leads to insulin resistance. Previous epidemiological studies did not examine the combined effects of dietary Ca, Mg, and vitamin D as well as ratio of Ca to Mg with T2DM. Therefore, we assessed the relationship between dietary intakes of Mg, Ca, and vitamin D  individually and in composite and T2DM in the National Health and Nutrition Examination Survey 2007–2014, which involved 20,480 adults  with comprehensive information on related nutrients, and anthropometric, demographic, and biomarker variables using multivariable logistic regression. The results indicated that dietary calcium at Q3 (812 mg/day) was significantly linked with T2DM in women . Dietary vitamin D at Q3 (5.25 μg/day) significantly reduced the odds of T2DM by 21% in men . This is an interesting study that has important implications for dietary recommendations. It is concluded that US adults having dietary Ca below the RDA were associated with increased risk of T2DM in all population and women, while higher ratio of Ca to Mg was associated with increased risk of T2DM in all population and increased vitamin D intake is related to decreased risk of T2DM in men. Moreover, further research is needed to make more definitive nutritional recommendations. This is an interesting study that has important implications for dietary recommendations. It is concluded that dietary calcium intake level of 812 mg/day (Q3) will increase the risk of developing T2DM in the general population and women. Additionally, the findings conclude that US adults having dietary calcium below the RDA were associated with increased risk of T2DM in all population and women, while higher ratio of Ca to Mg was associated with increased risk of T2DM in all population and increased vitamin D intake was associated with decreased risk of T2DM in men. Moreover, the conclusions should probably call for more studies to confirm these results in other settings. NHANES is a consecutive survey conducted every two years, representing one cycle of the US civilian noninstitutionalized population, and NHANES data set is publically available at 2.2n = 40,617) having age ≥ 18 years , whereas we excluded those participants having age < 18 years , pregnant women (n = 180), missing value daily energy intake , daily energy intake < 500 kcal/day (n = 256) and > 4,500 kcal/day (n = 357), missing value of body mass index , and the missing value of dietary intakes of calcium, magnesium, and vitamin D . The detailed descriptions are also provided in Figure n = 3,432; nondiabetes n = 17,048).We included the adult's participants’ NHANES 2007–2014 data sets from mobile examination center (MEC)  will be counted as cases, having hemoglobin A1C ≥ 6.5% and/or fasting glucose ≥ 7 mmol/L and/or having diagnosed T2DM.2.4The multipass approach method was used to measure dietary intake of magnesium, vitamin D, and calcium during in‐person 24‐hr dietary recall  and Quintile 4 as reference (model 4–6) by using logistic regression models, p‐value .05. Furthermore, we also stratified our models by sex and evaluated the intake of calcium and magnesium based on RDA (further down RDA or meeting the RDA based on sex‐ and age‐specific recommendations) for individual nutrient , educational level, exercise regularly (yes/no), current smoker (yes/no), current drinker (yes/no), annual household income, energy intake in groups, total vitamin D (mg/day) , or total Ca:Mg ratio intake (mg/day) (for the model including vitamin D quintile). Model 3 was further additionally adjusted for total calcium (mg/day) (model including magnesium quintile), total calcium (mg/day) (model including magnesium quintile).Model 1 was accustomed to sex, race, and age. Model 2 was moreover adjusted for body mass index groups  and noncases  , education level, exercise, smoking, drinking, annual household income, energy groups, and positive association observed at quintile 3 intake (Q3 812 mg/day) . A positive association was remained at quintile 3, after further adjustment for total magnesium intake . A significant inverse association remained between highest (median: 445 mg/day) versus. lowest quintiles (median: 152 mg/day) of magnesium and T2DM after alteration for race, sex, and age , and such inverse association did not remain statistically significant for model 2: OR: 1.03 95% CI  and model 3: OR: 0.99; 95% CI . We found a counter‐relationship among the highest quintile (445 mg/day) and magnesium versus the lowest quintile and T2DM (data not shown) when energy intake was excluded from our statistical models. The results were as follows: model 2: OR: 0.76; 95% CI: 0.60, 0.96, and model 3: OR: 0.76; 95% CI: 0.59, 0.99. We found a positive association of vitamin D at quintile 2 and T2DM when compared to Q4 reference in model 4; OR: 1.43; 95% CI: 1.14, 1.74, and such association remained at quintile 2 for model 5: OR: 1.29; 95% CI: 1.04, 1.59, and model 6: OR: 1.52; 95% CI: 1.14, 2.04. We found a significant positive association between highest (median: 4.98 mg/day) versus lowest quintiles (median: 1.76 mg/day) of calcium‐to‐magnesium ratio and T2DM after adjustment for model 1: OR: 1.23; 95% CI: 1.03, 1.48, P trend: 0.016, and such positive association remained after further adjustment for model 2: OR: 1.29; 95% CI: 1.05, 1.59, p trend: .010). Table 2), education level, exercise, smoking, drinking, annual household income, Energy‐Groups which reveals the positive association at quintile 3 (812mg/d) the value of model 2: OR 1.29; 95% CI: 1.01, 1.63 that shows 1.29 times more odds of T2DM, when the model was additionally adjusted for magnesium intake such association at quintile 3 remained/ retained .Table p trend: .00). Results showed a significant inverse association in women and men between highest (median: 431 mg/day) versus. lowest quintiles (median: 151 mg/day) of magnesium and T2DM after adjustment of age, sex, and race , while in men , respectively, such inverse association did not exist further in models 2 and 3. Dietary vitamin D in male has significantly reduced the odds of T2DM at quintile 4 (7.50 μg/day) , and we found that vitamin D intake at quintile 4 (7.50 μg/day) reduced the odds of T2DM in men , after further adjustment at quintile 3 (5.25 μg/d) , which shows inverse association to reduce the odds of T2DM. Furthermore, vitamin D intake at quintile (Q2) as compared to Q4 (reference) contains the recommended amount for model 4: OR: 1.61; 95% CI: 1.24, 2.09, model 5: OR: 1.53; 95% CI: 1.14, 2.04, and model 6: OR: 1.52; 95% CI: 1.14, 2.04, which revealed that T2DM participants at quintile 2 (3.5 μg/day) had more odds of T2DM when compared with reference Q4 (7.50 μg/day); however, no association was found for Ca: Mg ratio in men and women.A significant inverse association was found for women across dietary magnesium quintile and T2DM . It is concluded that dietary calcium intake of 812 mg/day (below RDA) increased the odds by 1.20 times, while the calcium‐to‐magnesium ratio between highest versus lowest quintiles increased the odds by 1.29 times in all populations. Calcium intake  increased the odds by 1.30 times in women, while vitamin D intake (5.25 μg/day) reduced the odds by 21% in men. Moreover, the importance of RDA indicated vitamin D intake when compared with the reference (Q4 recommended intake), and the odds of T2DM increased by 1.29 times in Q2 in both genders while 1.52 times in men.There was no relationship between the lowest versus highest quintiles of dietary intake of calcium, magnesium, and vitamin D and T2DM. Mostly, studies reported that one‐third of Americans did not meet adequate intake (AI) for calcium  as vitamin‐D facilities intestinal calcium absorption and insufficient calcium  because calcium is required in regulating glucose intolerance due to vitamin‐D deficiency in vivo , while one of the studies reported ethnic differences to exist among NHANES participants . Moreover, dietary supplement data inevitably have missing values which is why we do not include supplementation data. However, intake was done by the recall. Also, the results for the study may not be universal because the study was done in one geographic area; thus, further research is needed to make more definitive nutritional recommendations. Therefore, further prospective studies are warranted due to public health importance. Thus, essential micronutrients  related to chronic ailments of global concern include T2D as their public health importance.6This is an interesting study that has important implications for dietary recommendations. It is concluded that dietary calcium intake level of 812 mg/day (Q3) will raise the threat of developing T2DM in the general population and women. Additionally, the findings conclude that US adults having dietary calcium below the RDA were related to an enhanced risk of T2DM in all population, while higher ratio of Ca to Mg was associated with increased risk of T2DM in all population and increased vitamin D intake was associated with decreased risk of T2DM in men. Therefore, the conclusions should probably call for further studies to confirm these results in other settings.The authors declare no conflict of interest. “The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.”