Reactions of EBr3  with iPr2PAcenapLi  afforded the thermally stable peri-substitution supported donor–acceptor complexes, iPr2PAcenapEBr23 and 4. Both complexes show a strong P→E dative interaction, as observed by X-ray crystallography and 31P NMR spectroscopy. DFT calculations indicated the unusual As∙∙∙As contact (3.50 Å) observed in the solid state structure of 4 results from dispersion forces rather than metallic interactions. Incorporation of the excess AsBr3 in the crystal structure of 3 promotes the formation of the ion separated species [iPr2PAcenapAsBr]+Br− 5. A decomposition product 6 containing the rare [As6Br8]2– heterocubane dianion was isolated and characterised crystallographically. The reaction between iPr2PAcenapLi and EtAsI2 afforded tertiary arsine (BrAcenap)2AsEt 7, which was subsequently lithiated and reacted with PhPCl2 and Ph2PCl to afford cyclic PhP(Acenap)2AsEt 8 and acyclic EtAs(AcenapPPh2)2 9.A series of phosphorus-arsenic  This paper is dedicated to Prof Alex Slawin on the occasion of her 60th birthday, a world class crystallographer, a caring and compassionate colleague and mentor.3P) are known as prototypical neutral donors (Lewis bases) with the most common examples being their use as tunable ligands in coordination chemistry  Å, As1–Br2: 2.6400(18) [2.664(2)] Å . The P–Br and As–Br bond lengths in 3 and 4 indicate an intermediate character between ionic and covalent bonds; their elongation is rather significant when compared to the bond lengths in the covalent PBr5 (P–Br: 2.221 and 2.158 Å)  in 4). The P9 and C1 atoms occupy the equatorial positions, with the 3rd position taken up by the lone pair. The inequality and elongation of the phosphorus–halogen bonds are phenomena observed in all relevant structurally characterised phosphine–phosphine and phosphine–arsine donor–acceptor complexes, and presumably stem mainly from packing effects +Br−, co-crystallised with an AsBr3 molecule (see 3 molecules forming a centrosymmetric assembly. All Br∙∙∙As distances around the μ3-bridging Br2 atom (3.00–3.04 Å) are significantly elongated compared to all other As–Br bonds (2.3370(9)–2.4375(9) Å), supporting the interpretation of the bonding as ionic. Comparing the structures of 4 and 5 shows no significant change in the P–As bond length (2.3978(15) Å in 5). The most substantial changes are the shortening of the As1–Br1 bond to 2.4375(9) Å and the elongation of the other distance, As1···Br2, to 3.000(9) Å (c.f. As–Br bond lengths 2.634(2)–2.6639(19) Å in 4).Conducting the reaction of cule see , with tw5 from boiling acetonitrile, a small crop of colourless crystals was obtained. These were subjected to single-crystal X-ray diffraction and were found to be compound 6, consisting of a complex phosphonium cation and an unusual octabromohexaarsenate ([As6Br8]2–) dianion  Å), and an onset of 3-center 4-electron P···As–C interaction (P29···As1 3.145(4) Å; P29···As1–C1 angle is 176.9(3)°). A tentative mechanism of the cation formation involves an attack of the second molecule of iPr2PAcenapLi on 4 or 5. Only one previous incidence of crystallographic characterisation of octabromohexaarsenate dianion was found in the literature; this was in the form of its tetraphenylphosphonium salt 2– dianion. The reduction step may be analogous to that seen throughout the chemistry of pnictine–pnictine complexes, first described by Sisler [3PVCl]+Cl-), reducing itself to (AsIX)n. This proposed mechanism is distinct (but related) to that proposed by Macdonald for formation of octaiodocyclohexaarsenates, where the diphosphine ligand (L) cleavage from pre-assembled arsenic(I) species [LAs][I] was suggested , 344.04 (95) [M–2Br], 300.99 (90) [iPrPAcenapAs], 257.95 (95) [AcenapPAs], 234.74 (100) [AsBr2].Compound 2  in diethyl ether (40 mL), n-butyllithium  was added dropwise over 1 h and the mixture was left to stir for 2 h at the same temperature. The resulting suspension of iPr2PAcenapLi was added via cannula  over 1 h to a rapidly stirring solution of arsenic tribromide  in diethyl ether (80 mL), cooled to –78 °C. The reaction mixture was left to stir and warm to ambient temperature overnight. The suspension was filtered and the solid collected was washed with diethyl ether (30 mL). After drying in vacuo, 5 (contaminated with LiBr) was obtained as a pale-yellow powder . Crystals of 5 suitable for X-ray diffraction work were grown from THF at room temperature. Contamination with LiBr prevented satisfactory microanalysis and accurate determination of the yield. Solution NMR spectra of 5 are identical to those of 4.To a cooled (–78 °C) rapidly stirring solution of 6 suitable for diffraction work were obtained from recrystallisation of the crude product 5 from hot acetonitrile. No further characterisation was possible due to small amount available.Few crystals of n-butyllithium  was added dropwise to a rapidly stirred suspension of 5,6-dibromoacenaphthene 1  in THF (120 mL) at −78 °C. The mixture was maintained for 2 h at this temperature. To this, a solution of ethyldiiodoarsine  in THF (20 mL) was added dropwise over one hour at −78 °C. The resulting suspension was left to warm to room temperature overnight. The volatiles were removed in vacuo. Dichloromethane (50 mL) was added, and the resulting suspension was filtered. The product was obtained as a white powder after removal of the volatiles from the filtrate in vacuo. Recrystallisation of the crude material from dichloromethane gave 7 as colourless needle crystals , some of these were suitable for X-ray diffraction work. M. p. 199–200 °C. Elemental analysis: Calcd. (%) for C26H21AsBr2: C 54.96, H 3.72; found: C 54.86, H 3.65. 1H NMR: δH  7.60 , 7.48 , 7.08 , 7.00 , 3.29–3.19 , 2.04 , 1.19 . 131H} NMRC{: δC  146.2 , 145.7 , 140.7 , 135.6 , 134.6 , 133.0 , 132.3 , 119.4 , 119.2 , 115.5 , 29.1 , 28.8 , 23.0 , 10.4 . IR Data : ν = 3025 w (νArH), 2920 s, 2863 w (νCH), 1603 s, 1411 s, 1320 vs, 1251 m, 1200 m, 1100 m, 1044 m, 836 vs, 738 m, 602 m, 543 m. Raman  ν = 3063 m (νArH), 2952 m, 2932 s, (νCH), 1608 m, 1561 vs, 1442 vs, 1412 s, 1345 m, 1320 vs, 815 m, 702 s, 577 vs, 557 s, 290 vs. MS (ES+) m/z (%) 591 (100) [M + Na]. HRMS: m/z Calcd. for C26H21Br2AsNa (M + Na): 590.9103, found 590.9094.A solution of N,N,N′,N′–tetramethylethylene-1,2-diamine  was added to a suspension of 7  in THF (35 mL) and the mixture was cooled to –78 °C. A solution of n-butyllithium  was added dropwise with stirring over 1 h. The solution was maintained at –78 °C for 30 min before being warmed up and maintained at 0 to 10 °C for an hour. The solution was re-cooled to –78 °C and a solution of PhPCl2  in THF (5 mL) was added dropwise. The mixture was left to warm up to room temperature overnight with stirring. The volatiles were removed in vacuo and replaced with dichloromethane (30 mL) and the resulting suspension was filtered. Removal of the volatiles in vacuo once again gave 8 as a yellow solid . Crystals suitable for X-ray diffraction were grown from dichloromethane. Elemental analysis: The recrystallised material did not give satisfactory elemental analysis, presumably because of the contamination of the material with inorganic salts; the data fitted 8·1.45LiBr: calcd. (%) for C32H26AsP∙(LiBr)1.45: C 59.83, H 4.08; found: C 60.03, H 3.99. 1H NMR: δH  7.96 , 7.81–7.74 , 7.59 , 7.46–7.38 , 7.38–7.31 , 7.20 , 3.39–3.16 , 2.28 , 1.54 . 13C{1H} NMR was not acquired due to low solubility of 8. 31P{1H} NMR: δP  δP = –26.5 (s). IR : ν = 3071 w (νAr–H), 2962 s (νC–H), 1325 s, 1260 vs, 1095 vs, br, 1022 vs, 802 vs, 735 s, 701 m. Raman  ν = 3068 m (νAr–H), 2919 m (νC–H), 1603 m, 1583 vs, 1565 s, 1330 vs, 997 s, 537 s, 253 m. MS (CI+): m/z (%) 517.1 (58) [M + H], 487.1 (100) [M–Et]. HRMS (CI+): m/z Calcd. for C32H27AsP (M + H): 517.1066, found 517.1059.7  in THF (35 mL) and the mixture was cooled to –78 °C. A solution of n-butyllithium  was added dropwise with stirring over 1 h. The solution was maintained at –78 °C for 30 min before being warmed up and maintained at 0 to 10 °C for an hour. The solution was re-cooled to –78 °C and a solution of chlorodiphenylphosphine  in THF (5 mL) was added dropwise over 45 min. The solution was left to warm to room temperature overnight with stirring. The volatiles were removed in vacuo and replaced with dichloromethane (30 mL), and the resulting mixture was filtered. Removal of the volatiles in vacuo once again gave 9 as a pale yellow solid . Crystals suitable for X-ray diffraction were obtained in the form of monoxide 9a from dichloromethane. 1H NMR: δH  7.51 , 7.26–7.13 , 7.10–7.03 , 7.02–6.95 , 6.76 , 3.37–3.24 , 2.19 , 1.32 . 13C{1H} NMR: δC NMR was not acquired due to low solubility of 9. 31P{1H} NMR: δP  δP = –19.9 (s). IR : ν = 3044 m (νAr–H), 2962 vs, (νC–H), 1601 s, 1477 s, 1261 vs, 1020 vs, br, 800 vs, 693 vs. Raman  ν = 3047 vs, (νAr–H), 2930 s (νC–H), 1604 s, 1583 s, 1325 vs, 998 vs, 536 vs. MS (CI+): m/z (%) 779.1 (8) [M + H], 749.1 (16) [M–Et], 412.1 (32) [Ph2PAcenapAs]. HRMS (CI+) m/z Calcd. for C50H42P2As (M + H): 779.1978, found 779.1977.TMEDA  was added to a suspension of peri-substitution, novel dative complexes bearing pnictogen dibromide acceptor groups have been isolated and fully characterised. While their stability resembles that of their chlorine analogues, compounds 3 and 4 are thermally stable up to well above 100 °C and can be stored as solids under an inert atmosphere indefinitely, which makes them interesting for further synthetic use. Using excess AsBr3 gave species 5, in which a bromine atom of molecule 4 is ionically separated through interactions with co-crystallised molecules of AsBr3. The structures of 3–5 illustrate that in phosphine−pnictine complexes the local polarity (packing) effects within the crystal lattice result in rather different Pn−X bond distances due to the fine balance of the polar covalent and ionic bonding around the  negatively charged pnictoranido motifs. This is further corroborated by computational evaluation of the close As∙∙∙As interaction observed in 4, which is found to consist mostly of dispersion forces.Utilising 6 indicates disproportionationative redox instability of 5 at elevated temperatures, resulting in an unusual heterocubane dianion [As6Br8]2–, containing As(I) motifs.Isolation of compound 2 afforded no singly substituted species, only geminally disubstituted species 7. This compound has been shown to be a useful synthon, with the As–C bonds being tolerant to the strong base nBuLi. Thus, treatment of 7 with nBuLi proceeds through lithium-halogen exchange; subsequent reactions with chlorophosphine electrophiles afforded the new species, cyclic 8 and acyclic 9.Reaction of BrAcenapLi with EtAsI


Epidorylaimus procerus sp. n., collected from a natural habitat in Vietnam, is described and illustrated. It is distinguishable by its 2.16 to 2.46-mm-long body, lip region offset by depression and 15 to 17-µm broad, odontostyle 32 to 35-µm long, neck 415 to 461-µm long, pharyngeal expansion occupying 47 to 52% of the total neck length, uterus 76 to 130-µm long or 1.0 to 1.5-body diameters, vulva transverse (V = 40-43), caudal region conical elongate  with blister-like bodies, and hyaline portion occupying one-fourth its length, and males absent. Molecular analysis shows a close relationship of the new species and E. lugdunensis, supporting monophyly of the genus Epidorylaimus. Epidorylaimus Andrássy, 1986 is a widespread taxon in Dorylaimida. Originally proposed to accommodate 12 species previously included in Eudorylaimus Andrássy, 1959 its taxonomy was later updated by several authors . Morphometrics included Demanian ratios and other measurements. Microphotographs were taken with the same microscope provided with a Nikon Digital Sight DS-U1 camera. Raw photographs were edited using Adobe® Photoshop® CS.Specimens were measured, drawn, and identified with a Nikon Eclipse 80i light microscope equipped with differential interference contrast (DIC) optics and a drawing tube  and 2 µl of proteinase K (600 µg ml−1) (Thermo Scientific). The tubes were incubated at 65°C (1 hr) and then at 95°C (15 min). PCR was performed in a 30-µl final volume containing 24.9 µl of sterile water, 0.6 µl of each PCR primer, 0.6 µl of dNTP, 0.3 µl of Taq-polymerase, 3 µl of Buffer 10 x Thermo Scientific Green, and 1 µl of DNA-extracted solution. The PCR amplification profile consisted of 4 min at 94°C, 35 cycles of 1 min at 94°C, 1.5 min at 55°C, and 2 min at 72°C, followed by a final step of 10 min at 72°C. The primers used for amplification were D2A (5´-ACAAGTACCGTGAGGGAAAGTTA-3´) and D3B (5´-TCCTCGGAAGGAACCAGCTACTA-3´) for amplification of the D2-D3 region of 28S . Each nematode was transferred to a 0.5-ml Eppendorf tube containing 18 µl of Worm Lysis Buffer (WLB) , following the manufacturer’s manual. The sequencing reaction was performed with 15 ng of purified template, 4 µL of BigDye Terminator v3.1 Ready Reaction Mix, 2 µL of 5X Sequencing Buffer, and 3.2 pmol of forward/reverse primers for a total of 10-µL volume. The mixture was heated for 10 sec at 96°C, then 5 sec at 55°C, repeated for 32 cycles followed by 4 min at 60°C. The sequencing was performed on 3500 x L Genetic Analyzers  at the National Key Laboratory of Gene Technology . The sequences obtained were submitted to the GenBank database under accession numbers MT612084 and MT612088.© (Creative Suite 6).The obtained sequences were aligned with 35 other D2-D3 expansion segments of 28S rDNA gene sequences available in GenBank, using SeaView with the muscle algorithm followed by manual refining V4.5.3, . OutgrouEpidorylaimus procerus sp. n.In total, there were 15 females from one location in excellent state of preservation.Morphometricssee Table 1.a = 29-33) nematodes of medium size, 2.16-2.46-mm long. Their body was cylindrical, tapering anteriorly, extended posteriorly as an elongate, conical, and had a curved tail. Upon fixation, the habitus slightly curved ventrad, C- to hook-shaped. The cuticle was two-layered, 2 to 3-µm thick at the anterior region, 3.5 to 4 µm in mid-body, and 6.5 to 8.5 µm on the tail; the outer layer was thin and smooth, with constant thickness throughout the body; the inner layer was much thicker than the outer layer, which was especially conspicuous at the tail. The lateral chord was 15 to 19-µm broad, occupying 21 to 27% of mid-body diameter. Body pores were obscure.There were moderately slender  of neck length; gland nuclei are located as follows: DO = 56 to 63, DN = 59 to 65, S1N1 = 77 to 80, S1N2 = 78 to 83, and S2N = 89 to 90. The nerve ring was situated at 152 to 165 µm or 34 to 37% of the total neck length from the anterior end. The pharyngo-intestinal junction consisted of a well-developed, conical cardia measuring 19 to 29 × 11 to 15 µm, enveloped by the intestinal wall. The intestine lacked any relevant differentiation. The genital system was didelphic-amphidelphic, with both branches equally developed, anterior 212 to 278 µm or 10 to 13%, posterior 206 to 324 µm or 9 to 14% of the total body length. The ovaries were reflexed, the anterior measured 122 to 165 µm, and the posterior 140 to 229-µm long, often reaching and surpassing the oviduct-uterus junction, with oocytes arranged first in two or more rows and then in a single row. The oviduct was 91 to 132-µm long or 1.3 to 1.8-body-diameter long, consisting of a distal, slender section made of prismatic cells and a poorly developed proximal pars dilatata with visible lumen inside. A narrowing surrounded by a muscular ring (sphincter) separates the oviduct and the uterus. The uterus is a simple, tube-like structure measuring 76 to 130 µm or 1.0 to 1.5-body-diameter long. The vagina extended inward from 28 to 34 µm, occupying 39 to 46% of body diameter: pars proximalis 18-25 × 14-21 µm, and was surrounded by moderately developed circular musculature; pars refringens consisted of  two trapezoidal sclerotized pieces, 3.5-6 × 6-7 µm, with a combined width of 12.5 to 14.5 µm; pars distalis measured 4.5 to 6.5-µm long. Vulva was a transverse slit. Prerectum 2.0-3.4x, rectum 1.0-1.4x times the anal body diameter long. The tail was conically elongated, gradually tapering to a finely rounded tip, and strongly curved ventrad, with abundant saccate- or blister-like bodies inside the cuticle at its anterior third; two pairs of caudal pores, one subventral, another subdorsal, at about one anal-body diameter behind the level of the anus; the inner core could not reach the tail tip; therefore, a hyaline portion exists measuring 36 to 48 -µm long and occupying ca one-fourth (22-28%) of the total tail length.The lip region was truncate, offset by a distinct constriction, 1.9 to 2.3 times broader than high and one-fifth to one-fourth (21-26%) of body diameter at the neck base; the lips were amalgamated, with visibly protruding labial and cephalic papillae. Amphid fovea appeared to be cup-like, its aperture measured 4 to 5.5 µm, occupying up to one-third (28-33%) of lip-region diameter. Cheilostom was a truncate cone, without any particular differentiation. Odontostyle was relatively long and slender, 10.7 to 11.5 times longer than wide, length about twice (1.9-2.1 times) the diameter of the lip-region diameter, and about 1.38 to 1.51% of the total body length; the aperture measured 6.5 to 8.0-µm long, occupying up to one-fourth (20-25%) of odontostyle length. Odontophore was rod-like, nearly equal (0.8-1.0 times) in length to odontostyle. The pharynx was entirely and conspicuously muscular, with its slender portion enlarging gradually, and the basal expansion being 5.4 to 6.5 times as long as wide, 3.0 to 3.6 times longer than body diameter at the neck base, and occupying Unknown.Epidorylaimus spp. by a combination of the following character states and morphometrics: body 2.16 to 2.46-mm long, lip region offset by constriction, width 15 to 17-µm broad, odontostyle 32 to 35-µm long with aperture occupying 20 to 25% of its length, neck 415 to 461-µm long, pharyngeal expansion 194 to 238-µm long ; the female genital system was described as follows: didelphic-amphidelphic, uterus simple and 76 to 130-µm long (1.0-1.5 body diameters), and vulva transverse (V = 40-43); the caudal region was conically elongated  with blister-like bodies and hyaline portion occupying one-fourth its length.The new species is distinguished from other Epidorylaimus procerus sp. n. resembles E. mellenbachensis  Andrássy, 1986 and E. rivalis Gagarin, 1991., and can be easily distinguished from other species of the genus , less slender body (a = 29-33 vs 42-50), longer odontostyle , much more anterior vulva (V = 39-42 vs V = 50-55), and relatively longer tail . From the Russian species E. rivalis, it differs in being longer , the lip region is less differentiated (weak vs deep constriction), and it has a longer odontostyle (32-35 vs 28-30 µm). Males of E. rivalis are as abundant as females, whereas they are unknown in E. procerus n. sp. and thus likely to be rare or absent.E. lugdunensis de Man, 1880, the type species of the genus, supporting monophyly of Epidorylaimus. Both species appear closely related (100% support) to Crassolabium circuliferum Loof, 1961, a member of the family Dorylaimidae, and form part of a highly supported larger clade that includes members of the families Nordiidae and Qudsianematidae.Evolutionary relationships, as derived from the analysis of D2-D3 28S-rRNA gene sequences, are presented in a molecular tree . The newDipterocarpus sp. and Cinnamomum sp. as dominant plant species.Vietnam, Cao Bang Province, Cao Bang Natural Reserve , in a tropical evergreen forest soil with Female holotype and nine female paratypes were deposited with nematode collection of the University of Jaén, Spain. Five female paratypes were deposited with the nematode collection of the Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam.procerus = slim or svelte, and refers to the slender figure of these nematodes.The specific epithet is the Latin term


The nuclear receptors liver X receptor α (LXRα) and LXRβ are lipid sensors that regulate lipid metabolism and immunity. Natural killer T (NKT) cells, a T cell subset expressing surface markers of both natural killer cells and T lymphocytes and involved in antitumor immunity, are another abundant immune cell type in the liver. The potential function of the metabolic regulators LXRα/β in hepatic NKT cells remains unknown. In this study, we examined the role of LXRα and LXRβ in NKT cells using mice deficient for LXRα and/or LXRβ, and found that hepatic invariant NKT (iNKT) cells are drastically decreased in LXRα/β-KO mice. Cytokine production stimulated by the iNKT cell activator α-galactosylceramide was impaired in LXRα/β-KO hepatic mononuclear cells and in LXRα/β-KO mice. iNKT cell-mediated antitumor effect was also disturbed in LXRα/β-KO mice. LXRα/β-KO mice transplanted with wild-type bone marrow showed decreased iNKT cells in the liver and spleen. The thymus of LXRα/β-KO mice showed a decreased population of iNKT cells. In conclusion, LXRα and LXRβ are essential for NKT cell-mediated immunity, such as cytokine production and hepatic antitumor activity, and are involved in NKT cell development in immune tissues, such as the thymus. An understanding of the roles of hepatic immune cells is important to develop therapeutic and preventative strategies for liver diseases, such as nonalcoholic steatohepatitis and hepatocellular carcinoma2. NKT cells are a subset of T lymphocytes expressing surface markers common to NK cells and T lymphocytes, and recognize lipid antigens, such as α-galactosylceramide , which are presented with the major histocompatibility complex class I-like molecule CD1d on antigen-presenting cells3. Invariant NKT (iNKT) cells, which express a semi-invariant T cell receptor (TCR) containing Vα14-Jα18 and Vβ11 in human and Vα14-Jα18 and Vβ8.2, Vβ7 or Vβ2 in mice, are enriched in mouse liver (20–30% of hepatic lymphocytes) and possess a strong activity for the production of the Th1 cytokine interferon-γ (IFN-γ) and the Th2 cytokine interleukin-4 (IL-4)4. Hepatic iNKT cells are involved in inflammation, antitumor immunity, repair and regeneration in mice8. Impaired iNKT cell function is associated with several human cancers, including hepatocellular carcinoma9, and NKT cell therapy is a promising approach for antitumor immunotherapy, such as chimeric antigen receptor NKT cell therapy against neuroblastoma11. Although iNKT cells play an important role in hepatic immunity, the function of metabolism regulators in iNKT cells remains largely unknown.The liver is home to a variety of immune cells, including resident Kupffer cells, bone marrow-derived macrophages (BMDMs), natural killer (NK) cells, natural killer T (NKT) cells, T lymphocytes and B lymphocytes12. LXRα is abundantly expressed in liver, intestine, adipose tissue, kidney and macrophages, while LXRβ is ubiquitously present in the body. LXRs also regulate immune responses in immune cells, such as macrophages, through transrepression of other transcription factors, including nuclear factor κB and activation factor-1, and induction of genes involved in lipid metabolism, including the cholesterol efflux transporter ATP-binding cassette (ABC) transporter A1 (ABCA1)16. Recently, we found that LXRα and LXRβ are expressed in hepatic mononuclear cells (MNCs), including F4/80+ Kupffer cells/macrophages17. LXRα/β-knockout (KO) mice show increased numbers of F4/80loCD11b+ Kupffer cells/macrophages in the liver and enhanced inflammatory responses after treatment with lipopolysaccharide or CpG-DNA. In addition, high-fat and high-cholesterol diet feeding leads to the development of severe nonalcoholic steatohepatitis in LXRα-KO mice, associated with an increase in F4/80+CD68+CD11b+ Kupffer cells/macrophages18. Interestingly, high-fat and high-cholesterol diet-fed LXRα-KO mice have a decreased proportion of iNKT cells in the liver and are resistant to inflammatory responses to α-GalCer or concanavalin-A (Con-A) treatment. Thus, LXRα and LXRβ regulate immune responses mediated by iNKT cells in addition to Kupffer cells/macrophages in the liver. In this study, we investigated the role of LXRα and LXRβ in hepatic iNKT cell-mediated immunity using LXRα-KO, LXRβ-KO and LXRα/β-KO mice and found that both LXRs are necessary for iNKT cell development and related immune responses.The nuclear receptors liver X receptor α (LXRα) and LXRβ are transcription factors that are activated by oxysterols and regulate expression of genes involved in lipid metabolismloCD11b+ BMDMs are increased in the liver of LXRα/β-KO mice17. We examined the effect of deletion of LXRα and LXRβ on the population of other types of hepatic immune cells. The number of hepatic NKT cells (βTCR+NK1.1+) was decreased in LXRα-KO mice and LXRβ-KO mice compared to wild-type (WT) mice, and further lowered in LXRα/β-KO mice  was not affected by deletion of LXRα or LXRβ. Interestingly, iNKT cells (βTCR+CD1d-tetramer+) were drastically decreased in the liver of LXRα/β-KO mice, while the cell number was decreased to half and one-third of WT mice in LXRα-KO mice and LXRβ-KO mice, respectively  and stage 2 (CD44+NK1.1−) were comparable between WT and LXRα/β-KO mice, whereas stage 3 (CD44+NK1.1+) cells were significantly decreased in LXRα/β-KO mice. , Ifng (the gene encoding IFN-γ) and Tnf (the gene encoding tumor necrosis factor-α (TNF-α))  was unaffected and that of Nr1h2 (the gene encoding LXRβ) was slightly increased by α-GalCer treatment in WT cells. Interestingly, secreted IFN-γ protein levels were much higher in untreated LXRα/β-KO hepatic MNCs than in WT cells ) Fig. a. In con18. Plasma IL-4 levels at the 3-h time point were lower in LXRβ-KO mice and comparable to control levels in LXRα/β-KO mice  without α-GalCer treatment survived at 70 days after EL-4 cell injection  without α-GalCer treatment survived, and α-GalCer treatment did not increase the survival rate significantly (4 of 10 mice (40%); P > 0.05). Next, we analyzed metastatic tumors in the liver of α-GalCer-untreated mice 28 days after injection of EL-4 cells. All mice were alive at the 28-day time point  or BMDMs (F4/80loCD11b+) when comparing WT and LXRα/β-KO mice, while it was slightly decreased in resident Kupffer cells (F4/80hiCD11b−) from LXRα/β-KO mice  was not altered in LXRα/β-KO mice  was highly expressed compared to Nr1h3 (the gene encoding LXRα) , CD4+ T cells and CD8+ T cells in the thymus , stage 1 (CD24−NK1.1−CD44−), stage 2 (CD24−NK1.1−CD44+) and stage 3 (CD24−NK1.1+CD44+) in the thymus32. We evaluated the abundance of thymic iNKT cells at these developmental stages in adult mice. The distribution of abundance of iNKT cell stages in WT thymus was similar to that reported previously32. Compared to WT mice, stage 1, stage 2 and stage 3 cells were significantly decreased in LXRα/β-KO mice  Fig. c. mRNA lmus Fig. d. iNKT cice Fig. e. Stage loCD11b+ cells are increased in the liver of LXRα/β-KO mice17, iNKT cells were nearly absent in the liver and spleen of LXRα/β-KO mice  induced by the iNKT cell activator α-GalCer was impaired in LXRα/β-KO hepatic MNCs and in LXRα/β-KO mice Figs.  and 3, son Figs.  and 3. Incy Fig. . Treatme36. In contrast, LXRα activation inhibits dendritic cell-dependent antitumor responses37, although LXRα and LXRβ are necessary for dendritic cell chemotaxis39. Thus, LXRs function in antitumor immunity in an immune cell type-dependent manner. In contrast to the effect against NK cell-resistant EL-4 cells, antitumor effect against NK cell-sensitive Yac-1 cells was not repressed in LXRα/β-KO cells  was not altered in the thymus of LXRα/β-KO mice  and Con-A was from Vector Laboratories .Nr1h3-/- , Nr1h2-/-  and Nh1h3-/-; Nr1h2-/-  mice were kindly provided by Dr. David J. Mangelsdorf 51, and were backcrossed with C57BL/6 J mice for at least ten generations. Mice were maintained under controlled temperature (23 ± 1 °C) and humidity (45–55%) with a 12-h light, 12-h dark cycles, and with free access to water and chow . Young male mice (4 weeks old) and adult male mice (12–16 weeks old) were used for experiments. To activate NKT cells in vivo, mice were injected intravenously with α-GalCer (0.1 mg/kg of body weight) or Con-A (12.5 mg/kg of body weight) as reported previously18. Tissue samples were collected after euthanization with CO2 inhalation. All animal experiments were performed according to the protocols, which adhered to the Nihon University Rules concerning Animal Care and Use, approved by Nihon University Animal Care and Use Committee , and conformed to the ARRIVE guidelines.WT C57BL/6 J mice were purchased from Nihon CLEA , and CD45.1 (WT) B6-Ly5.1 mice were from Sankyo Labo Service Corporation, Inc., Tokyo, Japan. 18. For detection of lymphocytes including iNKT cells, MNCs were isolated without collagenase digestion. For detection of Kupffer cells and myeloid cells, MNCs were isolated with the 0.5 mg/mL collagenase digestion  for 25 min at 37 °C. F4/80+ cells, CD146+ cells and CD11c+ cells were isolated from the liver with collagenase perfusion using QuadroMACS sorting system  with Anti-F4/80 MicroBeads, CD146 (LSEC) MicroBeads and CD11c MicroBeads UltraPure (Miltenyi Biotec).MNCs were isolated with or without collagenase digestion and separated from parenchymal cells with Percoll solution  as reported previouslySplenocytes or thymocytes were isolated after filtration with 40 μm nylon mesh and removal of red blood cells. For NKT cell activation in vitro, cells were isolated with collagenase digestion and treated α-GalCer for 6 h for gene expression analysis, or 16 h for enzyme-linked immunosorbent assay.−NK1.1+) or NKT cells (βTCR+NK1.1+), cells were isolated without collagenase digestion, incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit  and anti-CD16/32 Fc blocker , and stained with fluorescein isothiocyanate (FITC)-conjugated anti-βTCR antibody , phycoerythrin (PE)-conjugated anti-NK1.1 antibody , biotin-conjugated anti-CD69 antibody  and PE-Cy5-streptavidin (Thermo Fisher Scientific).For detection of NK cells (βTCR+CD1d-tetramer+), cells were isolated without collagenase digestion, incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and anti-CD16/32 Fc blocker, and stained with FITC-conjugated anti-NK1.1 antibody , PE-conjugated CD1d tetramer complexed with α-GalCer , PE-Cy5-conjugated βTCR antibody , allophycocyanin-conjugated (APC-conjugated) anti-CD44 antibody  and/or Brilliant Violet 510-conjugated CD24 antibody .For detection of iNKT cells (βTCR−CD11b+), BMDMs (F4/80loCD11b+) and resident Kupffer cells (F4/80hiCD11b−), cells were isolated with collagenase digestion, incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and anti-CD16/32 Fc blocker, and stained with FITC-conjugated F4/80 antibody , Pacific Blue-conjugated anti-CD11b antibody  and APC-conjugated anti-CD1d antibody .For detection of CD1d-expressing antigen-presenting cells in hepatic MNCs such as neutrophils (F4/80+CD45.2−βTCR+CD1d tetramer+ cells).For bone marrow transplantation analysis, cells were isolated without collagenase digestion, incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and anti-CD16/32 Fc blocker, and stained with FITC-conjugated anti-NK1.1 antibody, PE-conjugated CD1d tetramer complexed with α-GalCer, PE-Cy5-conjugated βTCR antibody, APC-conjugated CD45.1 antibody  and Pacific Blue-conjugated anti-CD45.2 antibody  to identify WT-derived iNKT cells  and cell proportions were with FlowJo 10.4 software .+ βTCR+CD1d tetramer+ cells) or TECs (EpCAM+ cells). For the sorting of CD4+ T cells and CD8+ T cells, cells were incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and anti-CD16/32 Fc blocker, and stained with FITC-conjugated anti-βTCR antibody, PE-conjugated CD4 antibody , PE-Cy7-conjugated CD8a antibody  and APC-conjugated CD45 antibody  to identify CD4+ T cells (CD45+βTCR+CD4+ cells) and CD8+ T cells (CD45+βTCR+CD8a+ cells). Cells  were sorted using cell sorter , confirmed with the > 90% purity for each cell types and utilized for gene expression analysis.For the sorting of iNKT cells and TECs from WT mice, thymocytes were incubated with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and anti-CD16/32 Fc blocker, and stained with FITC-conjugated anti-βTCR antibody, PE-conjugated CD1d tetramer complexed with α-GalCer, and PE-Cy7-conjugated CD326 (EpCAM) antibody  and APC-conjugated CD45 antibody  to identify iNKT cells (CD45Cd1d1 (the gene encoding CD1d), 5′-AGG TCT GGG GAC AAT CTG AA-3′ and 5′-ATG GCT TCG TGA AGC TGA TG-3′; Cxcl16 (the gene encoding CXCL16), 5′-ACC AGT GGG TCC GTG AAC TA-3′ and 5′-CCT CAG GGG TCT GGG TAC TG-3′; Zbtb16 (the gene encoding PLZF), 5′-ACC ACA CGG GCT TGT GTA AA-3′ and 5′-ACG GTA CCT GGC AGC AAT AC-3′. The primers of Il4 (the gene encoding IL-4), Ifng (the gene encoding IFN-γ), Tnf (the gene encoding TNF-α), Nr1h3 (the gene encoding LXRα), Nr1h2 (the gene encoding LXRβ), Abca1 (the gene encoding ABCA1) and Ppib (the gene encoding Cyclophilin B) were reported previously54. The calculated RNA values were normalized with Ppib mRNA levels.Total RNA was isolated from hepatic MNCs, splenocytes or thymocytes with RNAiso Plus reagent , and cDNA was synthesized with ImProm-II reverse transcription system . Quantitative real-time polymerase chain reaction was performed with the StepOnePlus Real-time PCR system (Thermo Fisher Scientific) using Power SYBR Green PCR Master Mix reagent (Thermo Fisher Scientific). The mouse primer sequence was as follows: Plasma aspartate aminotransferase and alanine aminotransferase levels were measured with GOT·GPT CII-Test Wako . Plasma IL-4 and IFN-γ levels were measured with enzyme-linked immunosorbent assay kits .5 cells/20 g of body weight for the survival analysis or 1 × 106 cells/20 g of body weight for other analyses) were suspended in phosphate buffered saline and intravenously injected to mice. After 18 h, mice were injected intravenously with phosphate buffered saline or α-GalCer (0.1 mg/kg of body weight) and analyzed for survival rate. For other analyses, 28 days after EL-4 administration, hepatic tumor metastasis was counted, liver tissues were stained with hematoxylin and eosin, and plasma aspartate aminotransferase and alanine aminotransferase levels were measured with GOT·GPT CII-Test Wako.EL-4 mouse T lymphoma cells (5 × 106 cells per each well) were isolated by collagenase digestion and plated in a 96-well black plate. Target tumor cells (NK cell-resistant EL-4 or NK cell-sensitive Yac-1 cells)55 were labeled with 5 µg/mL calcein-AM (Sigma-Aldrich) and co-cultured with hepatic MNCs at the different concentration . After incubation for 4 h, fluorescent intensity  of culture media was measured with a FlexStation 3 microplate reader .Hepatic MNCs (5 × 107 cells per 20 g of body weight) isolated from donor B6-Ly5.1 mice. After 4 weeks, hepatic MNCs and splenocytes were isolated from recipient mice, and iNKT cell frequency was analyzed using FACSVerse flow cytometer.Mice were lethally irradiated (10 Gy) using MBR-1512R-3 , and injected intravenously with bone marrow cells .All data are indicated as means ± S.D. We performed one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons to analyze data of more than two groups, unpaired Student’s Supplementary Figures.


Caldicellulosiruptor, which are fermentative bacteria in the phylum Firmicutes, with 97.7–98.0% sequence identity to its closest relatives. Strain YA01 clearly exhibited N2-dependent growth at 70°C. We also confirmed N2-dependent growth in the relatives of strain YA01, Caldicellulosiruptor hydrothermalis 108 and Caldicellulosiruptor kronotskyensis 2002. The nitrogenase activities of these three strains were examined using the acetylene reduction assay. Similar activities were detected for all tested strains, and were slightly suppressed by the addition of ammonium. A genome analysis revealed that strain YA01, as well as other Caldicellulosiruptor, possessed a gene set for nitrogen fixation, but lacked the nifN gene, which encodes a nitrogenase iron-molybdenum cofactor biosynthesis protein that is commonly detected in nitrogen-fixing bacteria. The amino acid sequences of nitrogenase encoded by nifH, nifD, and nifK shared 92–98% similarity in Caldicellulosiruptor. A phylogenetic tree of concatenated NifHDK sequences showed that NifHDK of Caldicellulosiruptor was in the deepest clade. To the best of our knowledge, this is the first study to demonstrate the nitrogen-fixing ability of fermentative bacteria at 70°C. Caldicellulosiruptor may have retained an ancient nitrogen-fixing enzyme system.Fermentative nitrogen-fixing bacteria have not yet been examined in detail in thermal environments. In the present study, we isolated the thermophilic fermentative bacterium, strain YA01 from a hot spring. This strain grew at temperatures up to 78°C. A phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain YA01 belonged to the genus  According to a review by Bacteria and 1 phylum in Archaea from various environments  has been suggested to play an important role in nitrogen cycles in soil and animal intestines , a sulfidic and slightly alkaline hot spring, chemosynthetic microbial communities develop well at temperatures higher than 70°C , Nagano, Japan on January 8th, 2018. Hot spring water was slightly alkaline (pH 8.5–8.9) and contained 5.0–6.1 μmol L–1): 0.28 g K2HPO4, 0.053 g KH2PO4, 0.12 g MgSO4·7H2O, 0.125 g NaCl, 0.05 g yeast extract, 0.01 g CaCl2·2H2O, 2.5 mg FeSO4·7H2O, 2.5 mg MnSO4·5H2O, 2.5 mg Na2MoO4·2H2O, 2.5 g glucose, 2.5 g sucrose, and 2.5 g Na-pyruvate. The pH of the medium was adjusted to 7.5. Twenty milliliters of the medium was placed into a 70-mL glass vial. The vial was sealed with a butyl rubber stopper and aluminum cap, and then autoclaved after the gas phase had been replaced with N2. In total, 0.5 mL of the culture was repetitively sub-cultured every week in fresh medium. After 10 sub-cultivations, an isolate was obtained by the twice dilution-to-extinction technique. The single morphology of microbial cells was confirmed under a phase-contrast microscope .Winogradsky’s nitrogen-poor mineral medium  was prepCaldicellulosiruptor hydrothermalis 108, Caldicellulosiruptor bescii DSM 6725, and Caldicellulosiruptor kronotskyensis 2002 were obtained from DSMZ (Germany) (2:CO2 (8:2) gas phase in medium containing the following (L–1): 0.068 g KH2PO4, 0.087 g K2HPO4, 2.09 g MOPS, 0.33 g KCl, 0.25 g NH4Cl, 0.6 g MgSO4·7H2O, 0.4 g NaCl, 0.1 g CaCl2·2H2O, and 10 mL trace minerals. Twenty milliliters of the medium was prepared in 50-mL glass vials sealed with a butyl rubber stopper and aluminum cap. After autoclaving, 0.8 mL of a filter-sterilized 10% cellobiose solution, 0.2 mL of a 5% NaHCO3 solution, and 0.2 mL of a vitamin solution were injected into the vials. The trace minerals solution comprised the following (L–1): 1.5 g nitrilotriacetic acid, 3.0 g MgSO4·7H2O, 0.5 g MnSO4·5H2O, 1.0 g NaCl, 0.1 g FeSO4·7H2O, 0.1 g CaCl2·2H2O, 0.1 g CoCl2·6H2O, 0.13 g ZnCl2, 0.01 g CuSO4, 0.01 g AlK(SO4)·12H2O, 0.01 g H3BO3, 0.025 g Na2MoO4·2H2O, 0.024 g NiCl2·6H2O, and 0.025 g Na2WO4·H2O. The components of the vitamin solution were as follows (L–1): 2 mg biotin, 2 mg folic acid, 10 mg pyridoxine HCl, 5 mg riboflavin, 5 mg thiamine, 5 mg nicotinic acid, 5 mg pantothenic acid, 0.1 mg vitamin B12, p-aminobenzoic acid, and 5 mg thioctic acid.Bacterial cells were collected by centrifugation and total DNA was extracted according to a method reported by https://github.com/rrwick/Filtlong) with the parameters --min_length 1000 --target_bases 250000000, and processed error-prone reads using Canu v1.8  with ‘ggplot2’ in R package  (https://www.R-project.org/)  and sequenced by Bioengineering Lab. using the combination of DNBSEQ-G400 (MGI Tech) and GridION with the flow cell-type R9.4.1 (Oxford Nanopore Technologies) platform. Regarding DNBSEQ-G400, DNA was fragmented using a Covaris S2 ultrasonicator (Covaris) to obtain 500-bp DNA fragments. The DNBseq DNA library was prepared according to the manufacturer’s instructions and sequenced using DNBSEQ-G400 (pair-end 150-bp reads). In GridION, the library was prepared using the Ligation Sequence Kit (SQK-LSK109) after barcoding using Native Barcoding Expansion (Oxford Nanopore Technologies EXP-NBD104) and was then sequenced. In the DNBSEQ-G400 analysis, 3,500,000 read pairs (1.05 Gbp) were sampled using Seqkit (v. 0.11.0)  with theanu v1.8 . Proteinct.org/) .Rhodospirillum rubrum ATCC11170  was used as the outgroup  gas. In total, 0.5 mL of bacterial cultures pre-cultivated in nitrogen-poor medium were inoculated into fresh nitrogen-poor medium. To test the growth capability of C. bescii DSM 6725, a pre-cultivation was conducted using medium supplemented with 2 mmol L–1 of NH4Cl. Growth in the culture was assessed by measurements of optical density (OD) at 660 nm . Cultivation medium containing 2 mmol L–1 NH4Cl was also used to compare N2-dependent growth with growth on ammonium.Modified Winogradsky’s nitrogen-poor mineral medium (described above) was used to assess N2 gas atmosphere. At the exponential growth phase, a portion (0.5 mL) of the culture solution was removed and mixed with 0.05 mL of 10% Formalin Neutral Buffer Solution  to fix cells for the cell number count. The gas phase of culture vials was then replaced with N2 gas and 1.5 mL of 99.9999% acetylene gas was injected into each vial. Vials were incubated at 70°C and, after a 24-h incubation, 1 mL of 37% neutralized formaldehyde was added to stop the reaction. The production of ethylene by the reduction of acetylene was quantified using a GC-2014 gas chromatograph equipped with a flame ionization detector (Shimadzu) and 80/100 Porapak T (GL Science) column. Analysis conditions were as follows; carrier gas, N2 gas; column temperature, 70°C; injection temperature, 100°C; detector temperature, 100°C. Fresh medium containing no bacterial cells was prepared in the vial as a negative control to confirm abiotic ethylene production under the same conditions.Nitrogenase activity was detected using the acetylene reduction assay method (The 16S rRNA gene sequence was deposited in the DDBJ/EMBL/GenBank databases with the accession number LC603168. The accession numbers of the genomic sequences of strain YA01 were AP024480 (chromosome) and AP024481 and AP024482 (two plasmids).i.e., short rods  calculated using an ANI calculator (http://enve-omics.ce.gatech.edu/ani/)  ranged between 90.31 and 91.10%.DNBSEQ-G400 and GridION runs resulted in the generation of approximately 12,782,840 reads with a total of 1,917 Mbp and 155,919 reads with a total of 255 Mbp, respectively. The complete genome of strain YA01 consisted of a single chromosome with a length of 2,592,764 bp and two plasmids with lengths of 3,514 and 1,547 bp. The G+C content of the genome was 34.8%. Coding potential predictions identified 2,412 protein-coding genes, three rRNA operons, and 47 tRNA genes. Three 16S rRNA genes had the same sequence, which was identical to the 16S rRNA gene sequence amplified by PCR. Average nucleotide identity (ANI) between strain YA01 and its close relatives (1 and NifI2) , C. naganoensis NA10 (accession no. LACN01000001.1), C. danielii strain Wai35.B1 (accession no. LACM01000001.1), C. lactoaceticus 6A (accession no. CP003001.1), C. kronotskyensis 2002 (accession no. CP002330.1), C. kristjanssonii I77R1B (accession no. CP002326.1), C. hydrothermalis 108 (accession no. CP002219.1), and C. saccharolyticus DSM 8903 (accession no. CP000679.1) , two proteins involved in the biosynthesis of MoFe protein cofactors (NifB and NifE), and two proteins of the PII family involved in posttranslational nitrogenase regulation (NifId NifI2) . These s00679.1) . NifN wa00679.1) .Caldicellulosiruptor species possessing nitrogen fixation-related genes  and Hydrogenobacter sp. (Nif-B lineage) . A concatenated NifHDK phylogenetic tree was constructed for all ed genes . Strain er Nif-C . The Niflineage) . The theC. hydrothermalis 108, C. bescii DSM 6275, and C. kronotskyensis 2002, were cultivated in nitrogen-poor medium and ammonium-containing medium under the N2 or argon (Ar) gas phase . Acetylene-reducing activities were also observed under an incubation at 78°C for all strains: 9.50±5.05, 8.37±5.53, and 15.0±12.1 nmol C2H4 106 cells–1 24 h–1 by strain YA01, C. hydrothermalis 108, and C. kronotskyensis 2002, respectively. Activities at 78°C were weaker than those at 70°C.To test nitrogenase activity, strain YA01 and its relatives Caldicellulosiruptor and is closely related to C. hydrothermalis, C. bescii, and C. kronotskyensis. The results of the genomic analysis indicated that strain YA01 as well as C. hydrothermalis 108 and C. kronotskyensis 2002 possess a set of nitrogen fixation-related genes , which is required for the biosynthesis of the iron molybdenum co-factor of Mo-type nitrogenase   . Althougbacteria , most anCaldicellulosiruptor are frequently detected from microbial mats in geothermal springs , and anaerobic chemosynthesis   Nitrogen-fixing Ability and Nitrogen Fixation-related Genes of Thermophilic Fermentative Bacteria in the Genus https://doi.org/10.1264/jsme2.ME21018Supplementary Material


Some decrease in its macrocyclic cavity leads to a stacking interaction between the tricyclic fluorenone fragments. In the crystal, the fluorenonophane and chlorobenzene molecules are linked by weak C—H⋯π(ring) interactions and C—H⋯Cl hydrogen bonds.The molecular and crystal structures of the chlorobenzene solvate of fluorenonophane have been studied using X-ray diffraction analysis. The fluorenonophane contains two fluorenone fragments linked by two  9H,79H-3,5,9,11-tetraoxa-1,7-difluorena-4,10-dibenzenacyclododecaphane-19,79-dione (fluorenonophane), exists as a solvate with chlorobenzene, C42H28O6·C6H5Cl. The fluorenonophane contains two fluorenone fragments linked by two m-substituted benzene fragments. Some decrease in its macrocyclic cavity leads to a stacking interaction between the tricyclic fluorenone fragments. In the crystal, the fluorenonophane and chlorobenzene molecules are linked by weak C—H⋯π(ring) interactions and C—H⋯Cl hydrogen bonds. The Cl atom of chlorobenzene does not form a halogen bond. A Hirshfeld surface analysis and two-dimensional fingerprint plots were used to analyse the intermolecular contacts found in the crystal structure.The title compound, 1 In structure 3, the fluorenone fragments are oriented in the same directions (cis-orientation) while the orientation of these fragments is trans in structures 1 and 2. meta-Substitution of the two benzene fragments results in a smaller macrocycle cavity as compared to fluorenonophanes 1 and 2 with para-substituted benzene fragments. As a result, the two fluorenones are slightly bowed inwards . In contrast to structures 1 and 2, the macrocycle in structure 3 does not contain any molecules inside its cavity. Therefore, the structure under study is a chlorobenzene solvate of fluorenonophane.Fluorenonophane ts Fig. 1. The macA⋯Cg2 and C22—H22A⋯Cg1 interactions  was not found in the electrostatic potential map around the halogen atom  surface resolution and a fixed colour scale of −0.134 (red) to 1.206 (blue) were generated separately , then with water (3 × 50 mL) to a neutral pH. After drying over MgSO4, the CHCl3 was evaporated under reduced pressure. The product was purified by chromatography on silica gel (Acros 0.060 ÷ 1/5), eluent CHCl3–EtOH, 500:1. The yield of cyclophane 3 was 0.11 g (7.2%), m.p. >573 K, dec. 1H NMR (DMSO-d6), δ, p.p.m.: 5.25 s , 6.46–6.56 m , 7.04 t , 7.18 s , 7.57 m . MS: FAB, m/z 628 [M + H+]. Analysis calculated for C42H28O6: C, 80.24; H, 4.49. Found: C, 80.44; H, 4.76%. Crystals were obtained by crystallization of fluorenonophane 3 from chlorobenzene.A solution of 1.75 g (4.78 mmol) of 2,7-bis(bromomethyl)-9ed Fig. 5. The pre2 and refined as riding atoms with Uiso(H) = 1.2Ueq(C).Crystal data, data collection, and structure refinement details are summarized in Table 210.1107/S2056989021011865/mw2179sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021011865/mw2179Isup2.hklStructure factors: contains datablock(s) I. DOI: 2121128CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Background: Phillyrin (Phi) is the main polyphenolic compound found in Forsythia suspensa. Recent studies have revealed that Phi has potent antioxidative and anti-inflammatory effects. However, whether Phi could relieve blood–brain barrier (BBB) damage following traumatic brain injury (TBI) remains unknown.Materials and Methods: Lipopolysaccharide (LPS) was used to activate primary microglia, which were then treated with different doses of Phi or the peroxisome proliferator–activated receptor-gamma (PPARγ) antagonist (GW9662). CCK-8 assay was used for evaluating cell viability, and the cytokines , microglial phenotypic markers , PPARγ, and NF-κB were determined by RT-PCR, Western blot, or cellular immunofluorescence. Primary cultured mouse brain microvascular endothelial cells (BMECs) were stimulated by the condition medium (CM) from microglia. The cell viability, angiogenesis, and tight junction of BMECs were determined via CCK-8 assay, tube formation assay, and Western blot . Furthermore, the mouse TBI model was constructed and treated with Phi and/or GW9662. The BBB integrity was evaluated by H&E staining, Evans blue staining, and tissue immunofluorescence.Results: Phi markedly restrained the pro-inflammatory (“M1” state) cytokines and promoted anti-inflammatory (“M2” polarization) cytokines in LPS-mediated microglia. Phi mitigated “M1” polarization and promoted “M2” polarization of microglia via enhancing PPARγ and inhibiting the NF-κB pathway. The PPARγ antagonist GW9662 significantly repressed Phi-mediated anti-inflammatory effects. Meanwhile, Phi enhanced the viability, tube formation ability, and cell junction of BMECs. In the TBI mouse model, Phi promoted “M2” polarization, whereas it repressed the “M1” polarization of microglia. In addition, Phi reduced TBI-mediated BBB damage. However, the protective effects of Phi were reversed mainly by GW9662 treatment.Conclusion: Phi prevents BBB damage via inhibiting the neuroinflammation of microglia through the PPARγ/NF-κB pathway, which provides a potential therapeutic drug against TBI. Emerginvia transforming the “M2” polarization of microglia , microglia, have a pivotal function in both the injury and protective processes following TBI . Whethericroglia . Thus, iicroglia .Forsythia suspensa (Thunb.) Vahl, also known as Lianqiao in Chinese traditional medicine, is often used in clinics for its extensive pharmacological activities (Forsythia suspensa) relieves inflammatory cytokine expression and prevents the abnormal adhesion and migration of monocytes to type II alveolar epithelial cells via enhancing miR-124 , TBI group (n = 20), TBI+Phi group (n = 20), and TBI+Phi+GW9662 group (n = 20). DMSO was used for dissolving Phi and GW9662 , which were diluted with 0.9% saline. Phi (10 mg/kg) and/or GW9662 (1 μmol/kg) was given immediately 1 h before surgery and after that daily for seven days by intraperitoneal injection as referred to in a previous study  weighing 20–22 g were obtained from the Animal Center of Tongji Medical College of Huazhong University of Science and Technology. Those mice were fed under specific pathogen-free (SPF) conditions and had access to a standard diet. When the mice were accustomed to the living environment, they were randomly divided into four groups: the Sham group ]. The TBI model induced by controlled cortical impact (CCI) was conducted as previously described . After svia the tail vein. Two hours later, the mice were sacrificed and perfused with saline to remove the residual dye from the vessels. Next, the hemispheres of the mice were taken, and methanamide (Sigma-Aldrich) was used to incubate the tissues. After that, the percentage of the EB-stained brain volume was calculated . In addition to that, the EB content of the hemispheres was tested by a trichloroacetic acid solution at 620 nm. The EB content was counted as intensity/mg of brain tissue.The Evans blue dye was used for the evaluation of BBB integrity . In briePrimary microglia were obtained from C57BL/6 mice (1–2 days after birth), referring to our previous study . The scaPrimary brain microvascular endothelial cells (BMECs) were obtained from 2-week-old C57BL/6 mice as described previously . In brie5 cells per well. Twelve h after seeding, the microglia were treated with LPS (10 ng/ml), Phi (0–40 μg/ml), or GW9662 (1 μM) for 4 h. Next, the culture medium was collected, and a new fresh complete medium was added. Centrifugation  was used for removing the cell debris in the culture medium, which was then treated with primary BMECs seeded in 24-well plates (1 × 105 cells per well) for 12 h. Then, the culture medium of BMECs was removed and supplemented with a new fresh culture medium. After another 24 h of culture, the culture medium of BMECs was collected for further experiments.Primary microglia were cultured on 6-well plates with 5 × 10After treating the condition medium of microglia, the culture supernatant from BMECs was collected. The level of VEGFA  and EGF  was determined using the ELISA kit according to the manufacturer’s protocols. The experiment was repeated five times.The microglia, BMECs, and brain tissues were collected, and the total proteins were separated by RIPA . The protein concentrations were determined using a BSA Kit . Next, the total proteins were isolated by SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk for 1 h at room temperature and then incubated with primary antibodies, including anti-MMP3 , anti-MMP9 , anti-iNOS , anti-COX2 , anti-CD86 , anti-Arg1 , anti-Ym1 , anti-CD206 , anti-CD206 , anti-PPARγ , anti–phospho-NK-κB , anti–NK-κB , anti-occludin , anti-ZO1 , and anti–claudin-5  at 4°C overnight. Next, the membranes were incubated with peroxidase-conjugated goat anti-mouse IgG secondary or peroxidase-conjugated goat anti-rabbit IgG  for 1 h at room temperature. Finally, the brands were exposed and photographed using a Gene Gnome exposure instrument. β-actin  was used as the internal reference of the other proteins. The experiment was repeated thrice.The brain tissues of TBI mice on the 7th day were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and dissected into sections with 10 μm thickness. Each section was then deparaffinized, hydrated, washed, and stained with hematoxylin–eosin (H&E) using a commercially purchased kit .The prepared brain sections or cells were permeabilized with Triton X-100 and blocked using goat serum. Then the sections or cells were incubated with primary antibodies including anti-occludin , anti–p-NF-κB , anti–Iba-1 , anti–ZO-1 , anti-PPARγ , anti-iNOS , anti-Arg1 , and anti–claudin-5  overnight at 4°C. On the next morning, the sections or cells were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to Alexa 488 or Alexa 647  for 1 h at 37°C. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) . After mounting, the immunofluorescent or immunohistochemical signals were observed under an Olympus microscope . The positive cells were counted using Image J, with the researcher being blinded to the treatment conditions.The prepared brain sections were permeabilized with Triton X-100 and blocked with goat serum. Permeabilization and blocking were omitted for IgG immunostaining. The sections were incubated with primary antibodies of anti–caspase-3  overnight at 4°C. Then, the sections were incubated with the secondary antibody at room temperature for 2 h. After that, 3,3′-diaminobenzidine (DAB) chromogen was used to develop the peroxidase activity, and the nuclei were stained using 0.02% Mayer’s hematoxylin. Finally, the sections were observed under a camera-coupled bright-field microscope.Total RNA from the cells or tissues was isolated using TRIzol . Next, the total RNA was reverse-transcribed to cDNA with the PrimeScriptTM RT Reagent Kit  according to the manufacturer's instructions. The produced cDNA was amplified by quantitative real-time PCR on an ABI-Prism 7500 Real-Time PCR System  using SYBR Premix Ex TaqTM II (Takara). GAPDH was used as the internal control of the detected genes. The primer sequences used in this study were as follows: IL-1β, forward, 5′-ggctcatctgggatcctctc-3′, reverse, 5′-tcatcttttggggtccgtca-3′; iNOS, forward, 5′-gtttgaccagaggacccaga-3′, reverse, 5′-gtgagctggtaggttcctgt-3′; COX2, forward, 5′-ccccaaacacagtgcactac-3′, reverse, 5′-agaggttggagaaggcttcc-3′; CD86, forward, 5′-gcacgtctaagcaaggtcac-3′, reverse, 5′-catatgccacacaccatccg-3′; IL-6, forward, 5′-ggagcccaccaagaacgata-3′, reverse, 5′-caggtctgttgggagtggta-3′; TNF-α, forward, 5′-ggattatggctcagggtcca-3′, reverse, 5′-acattcgaggctccagtgaa-3′; IL-4, forward, 5′-tggtgttcttcgttgctgtg-3′, reverse, 5′-acctggtagaagtgatgccc-3′; IL-10, forward, 5′-acaccttggtcttggagctt-3′, reverse, 5′-tcgctttgtacaacagcacc-3′; VEGFA, forward, 5′-gacatcctcctcccaacaca-3′, reverse, 5′-attacctggatgccgcaaac-3′, EGF, forward, 5′-cggaagcagctatcaaaccc-3′, reverse, 5′-gagacaggccagcatctact-3′, TGF-β, forward, 5′-actgcttcccgaatgtctga-3′, reverse, 5′-ttcctgtagacacacccacc-3′; Arg1, forward, 5′-gctgggaaggaagaaaaggc-3′, reverse, 5′-tgccgtgttcacagtactct-3′; Ym1, forward, 5′-ctcaacctggactggcagta-3′, reverse, 5′-ctgctcctgtggaagtgagt-3′; CD206, forward, 5′-aaggaaaccatggacaacgc-3′, reverse, 5′-actttgctcccatccatcca-3′; PPARγ, forward, 5′-aactccctcatggccattga-3′, reverse, 5′-gcattgtgagacatccccac-3′; and GAPDH, forward, 5′-aacgaccccttcattgacct-3′, reverse, 5′-atgttagtggggtctcgctc-3′. The experiment was repeated thrice.3 cells per well. Twelve hours after seeding, the microglia were treated with LPS (10 ng/ml), Phi (0–40 μg/ml), or GW9662 (1 μM) for 4 h. After that, 10 μl of the CCK-8 solution was added into each well, and the cells were incubated for 1 h at 37°C. Absorbance measurement at 450 nm was performed on the Thermo Scientific microplate reader. The value was used to calculate cell viability by setting the control as 100%. The experiment was repeated thrice.Cell Counting Kit 8 (CCK-8) assay was used to evaluate the viability of microglia or BMECs using the CCK-8 kit . Primary microglia or BMECs were cultured on 96-well plates with 5 × 104 cells/well) on the surface of the solidified Matrigel matrix and incubated for 12 h at 37°C. A light microscope  was used for observing capillary tube formation. The length of the tubes was counted using ImageJ software .The capillary tube formation ability of BMECs was measured using Matrigel matrix . Matrigel matrix (50 μl in each well) was pre-coated on the 96-well plates 12 h before cell seeding, and the plates were put in the refrigerator at 4°C. After being treated with the condition medium from microglia for 12 h, the BMECs were collected and seeded  was used for statistical analyses. All data are expressed as mean ± standard deviation (SD). Multiple comparisons were analyzed by one-way ANOVA followed by a Primary cultured microglia were treated with different doses of Phi (0–40 μg/ml) or LPS (10 ng/ml). CCK-8 assay was performed to test the viability of microglia. The obtained data showed that all concentrations of Phi (ranging from 10 to 40 μg/ml) had no adverse effects on the viability of the microglial cells at 4 and 24 h with or without the insult of LPS . Then, win vitro, we first performed RT-PCR and Western blot to detect the PPARγ expression in microglia. As the data showed, Phi enhanced the mRNA and protein levels of PPARγ, whereas LPS reduced PPARγ mRNA and protein levels , a PPARγ antagonist. We then treated microglia with Phi (40 μg/ml) or LPS (10 ng/ml). After that, RT-PCR and Western blot were conducted to determine the inflammatory reactions of microglia. As the data showed, GW9662 treatment promoted the expression of “M1” markers in microglia, including IL-1β, IL-6, TNFα, iNOS, COX2, and CD86, whereas it inhibited the expressions of “M2” markers, including IL-4, IL-10, Arg1, Ym1, and CD206 , and/or GW9662. The cell viability and tube formation ability of BMECs were repressed by LPS-CM stimulation. Phi treatment in BMECs significantly enhanced BMEC cell viability and tube formation ability (compared with the LPS-CM group), whereas GW9662 treatment mostly reversed Phi-mediated effects , also known as Lianqiao, is often used for treating influenza and upper respiratory tract infection in combination with other Chinese herbal preparations (Forsythia suspensa (Thunb.) fruits or leaves have antioxidative and anti-inflammatory effects  by increasing the levels of 2-arachidonoylglycerol (2-AG)  rats subjected to cerebral ischemia–reperfusion (I/R) injury (Forsythia suspensa (Thunb.) have potential effects in treating central nervous system diseases.In Chinese traditional medicine, arations . Many bi effects . For exa effects . An in vl (2-AG) . Rutin, ) injury . TherefoForsythia suspensa (Thunb.), Phi has been found to express multiple biological functions, such as improving insulin resistance /CD16(+) M1 microglia and increasing the number of Iba1(+)/CD206(+) M2 microglia after stroke  often causes damage and leakage after TBI, resulting in increased extravasation of immune cells and enhanced secondary injury . Recentlvia promoting the microglial “M2” polarization through the PPARγ/NF-κB pathway, and Phi relieves TBI-induced BBB injury caused by microglia. Further research work exploring how Phi modulates the PPARγ expression in the mouse TBI brain lesions or microglia is needed, which will help to delineate more clearly the potential clinical usage of Phi.In summary, the main findings of this study are that Phi has anti-inflammatory effects


Euphorbia grandicornis is widely utilized in traditional medicine for the treatment of microbial infections including sexually transmitted diseases such as syphilis, gonorrhoea and for healing of wounds.Euphorbia grandicornis chemical constituents.The aim of this work was to isolate and evaluate the antibacterial and anticancer activities of Chemical constituents were isolated and identified using various spectroscopic techniques such as IR, MS, and NMR. The single point growth inhibitory potential of the compounds was determined using a 96-well plate based assay.2Cl2 crude extracts exhibited potent antibacterial activity against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538 with percentage growth of 94.90 ± 4.24 and 29.47 ± 4.89 respectively. Hence, the CH2Cl2 crude extract was further subjected to column chromatography which resulted in the isolation of methyl 2,5-dihydroxybenzoate (1), n-octyl benzoate (2), friedelanol (3), and germanicol (4) and identification of compounds 12–24 for the first time in the species based on the LC-MS/MS spectroscopic data. The purified compounds (1–4), and previously reported compounds (5–11) were evaluated for antibacterial activities against S. aureus and E. coli, as well as the cytotoxicity effects against HeLa cells. Of the purified compounds, methyl 2,5-dihydroxybenzoate (1), was the most active against E.coli and S. aureus with a percentage growth of 19.12 ± 0.65 and 23.32 ± 0.23 respectively. β-amyrin (6), and β-sitosterol (8), were active against S. aureus with percentage growth of 27.17 ± 0.07, and 47.79 ± 2.99 respectively.The CHE. grandicornis, is a rich source of chemical constituents that may provide new lead compounds for the development of antibacterial agents.The results obtained from this study indicate that The online version contains supplementary material available at 10.1186/s12906-022-03571-8. Staphylococcus aureus and Escherichia coli are involved in the many respiratory and skin infections, which cause urogenital, gastrointestinal, and wound contamination, are some of the resistant strains to most of the available antibiotics D25–75.53  at 20 °C. The HR-ESI-MS exhibited a pseudo-molecular ion [M + H]+ at m/z 169.0107 which indicated a molecular formula C8H8O4 calculated for m/z 168.0423. The IR spectrum displayed absorptions bands at 2957.8 cm− 1 which revealed the presence of C = C and absorptions at 1214 cm− 1 due to C-O stretches of the methoxy group. Broad absorption bands at 3244 cm− 1 were attributed to the hydroxyl groups (OH). The 1H NMR (400 MHz) displayed a characteristic singlet peak of a methoxy with proton resonance at δH 3.94  ppm, integrating to three protons. The spectrum also displayed aromatic protons at δH 6.93  and 7.29 7.41  ppm. The 13C NMR spectrum showed eight carbon resonances at δC 122.9 (C-1), 156.7 (C-2), 114.8 (C-3), and 118.5 (C-4) ppm. The spectrum also displayed a carbonyl resonance at δC 167.4 and a methoxy carbon resonance at δC 52.4 ppm. The NMR spectral data compared well with a known compound named; methyl 2,5-dihydroxybenzoate, previously isolated from Illicium dunnianum as in Tables SMethyl 2,5-dihydroxybenzoate 1) was obtained from the CH was obtan-Octylbenzoate, (2) was obtained from the CH2Cl2 extracts of E. grandicornis as a pure white powder. The melting point was determined between 75 and 95 °C with its optical specific rotation recorded as [α]D + 54.10  at 25 °C. The HR-ESI-MS spectrum showed a pseudo-molecular ion [M + H]+ at m/z 235.0723, calculated for C15H22O2, m/z 234.1620. The fragmentation patterns were consistent with that of a monosubstituted benzoate ester with a molecular peak at [M + H]+ = 116.1248 (C8H17)+ due to an octyl side-chain molecular ion, and at [M-H]+m/z 123.0574 (C7H6O2)+ due to benzoic acid molecular ion. The IR spectrum exhibited an absorptions band at 1718 cm− 1 which revealed the presence of a carbonyl group, absorption band at 1107 cm− 1 due to C-O stretches and more absorption bands at 2913 cm− 1 and 2860 cm− 1 attributed to C-H  stretches.1H NMR (400 MHz) displayed characteristic signals of a benzoate ester with proton resonances at δH 8.02 , 7.52  and 7.44  ppm. The spectrum exhibited a triplet proton resonance at δH 4.31  ppm. In addition, an upfield triplet proton resonance at δH 0.83  ppm was attributed to the terminal methyl group, while a broad clustered proton resonance at δH 1.22 ppm accounted for twelve protons revealing the presence of six methylene groups. The 13C NMR spectrum displayed carbon resonances typical of a benzoate ester  D25 + 34.20 . The HR-ESI-MS spectrum for this compound displayed a pseudo-molecular ion [M + H]+ at m/z 429.1763 indicating a molecular formula C30H52O calculated for m/z 428.7333. The IR spectrum exhibited absorption bands at 3340 cm− 1 due to the hydroxyl group and bands at 2925 and 2866 cm− 1 were ascribed to C-H stretches. Analysis of the 1H NMR (400 MHz) spectra displayed resonances due to an oxymethine proton at δH 3.66  ppm which correlated with a carbon resonance at δC 72.8 (C-3) ppm as observed in the HSQC spectrum. Other observed methyl proton resonances were δH 0.97 , 1.11 , 0.93 , 0.97 , 1.01 , 1.00 , 1.17  and 0.89  ppm. Based on the NMR data and by comparison to literature values as shown in Tables Sβ-friedelanol (3) . These values were consistent with the theoretical molecular mass of kaempferol-O-rutinoside (19) and hence the peak was assigned to the known compound − at m/z 447.0910 [LC-MS3)] with an MS fragmentation ion at m/z 285.0367 and which was consistent with the reference standard, was assigned as kaempferol-O-glucoside (18) [The LC-MS chromatogram displayed a major peak [M-H]compound , 32. Theide (18) . These f−at m/z 463.1227 [LC-MS4)] was assigned as quercetin-O-hexoside (23). The LC-MS spectral data of quercetin-O-hexoside agreed with the previously reported quercetin hexoside by Hussein et al. [− at m/z 289.0690 with a fragmentation molecular ion at m/z 230.1216 and m/z 279.1215 and retention time 17.13 min [LC-MS6)] was assigned as (+)-catechin (15) previously identified in Argan fruits. The fragmentation pattern was observed, and its retention time was comparable with the reference standard and with the theoretical data for catechin. The observed peak [M + H]+ at m/z 449.2012 and the fragmentation patterns n et al. . Moreovethyme by  and iden+ at m/z 697.1594 (for C30H32O19) with a retention time of 16.26 min [LC-MS5)] was attributed to a flavonoid glycoside kaempferol 3-O--β-glucoside (17), a rhamnosyl flavonoid previously isolated from Clitoria termatea termatea . The fra2Cl2 extract had antibacterial activities against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538 with a percentage growth of 94.90 ± 4.24 and 29.47 ± 4.89 μg/mL respectively. Table S. aureus, E. coli, and HeLa cells on exposure to crude extracts and pure compounds.This study revealed that the CHS. aureus indicating selective activity with a percentage growth of 94.90 ± 4.24 and 29.47 ± 4.89 against E. coli and S. aureus respectively. The results are in agreement with the previous studies which showed significant antibacterial activities of E. royleana hexane extracts [Bacillus subtilis [E. guyoniana showed significant activities against strains used with percentage growth values ranging from 1.47 to 61.78 mg/mL in the order of Staphylococcus aureus > Streptococcus faecalis > Escherichia coli [The extract was more active against extracts . The extsubtilis . Antibachia coli .1), was the most active against E.coli and S. aureus with percentage growth values of 19.12 ± 0.65 and 23.32 ± 0.23 μg/mL respectively. β-amyrin (6), and β-sitosterol (8), were active against S. aureus with percentage growth values of 27.17 ± 0.07, and 47.79 ± 2.99 μg/mL respectively. Other compounds exhibited some antibacterial activities with percentage growth values ranging from 51.94 ± 15.59 to 83.57 ± 4.45 for S. aureus and from 79.10 ± 1.36 to 132.55 ± 2.16 for E. coli as presented in Table Among the tested compounds, Methyl 2,5-dihydroxybenzoate (7), (24R)-tirucalla-8,25-diene-3β, 24-diol (9), and euphorbol (10) exhibited cytotoxic activities against HeLa cells with values of 15.24 ± 0.26, 18.81 ± 2.02 and 50.57 ± 11.29 respectively, as illustrated in Fig. It was interesting to note that stigmasterol , S. brasiliensis (UFPE 121), and E. coli (ATCC 8739), with minimum inhibition concentration (MIC) values ranging from 256 to 1024 μg/mL. Of the tested compounds, ampelopsin showed a MIC value of 16 μg/mL against E. coli (ATCC 8739), compared to tetracycline  [E. royleana displayed significant antimicrobial activities [In related studies, evaluation of compounds isolated from i ATCC 87, with mitivities .Euphorbia grandicornis Blanc crude extracts afforded twenty-four compounds (1-24). Compounds (1-4) were reported for the first time from the species. Methyl 2,5-dihydroxybenzoate (1) displayed good antibacterial activities against E. coli and S. aureus with a percentage growth of 19.12 and 23.32 at 50 μg/mL respectively. The results obtained from this study shows that E. grandicornis, is rich source of chemical constituents that may provide new lead compounds for the development of antibacterial agents.Chromatographic analysis of Additional file 1: Supplementary Materials. The following are available online alongside Appendix 1–12, Appendix LC-MS1-LCMS6, Table S1.13C (δc) (100.6 MHZ) NMR data for compounds 1-11 in CDCl3, Table S2.1H  NMR (400 MHZ) data for compounds 1–4 in CDCl3.


Cytotoxic assay analyzed percent killing of leukemia cell lines due to the effect of γδ T cells alone or in combination with α-AAA-A or α-AAA-B. Synthesized biologically active molecule α-AAA-A exhibited increased cytotoxicity of HL-60 (54%) and K562 (44%) compared with α-AAA-B (44% and 36% respectively). Similarly, α-AAA-A showed low IC50 values for HL-60 (1.61 ± 0.11 μM) and K562 (3.01 ± 0.14 μM) compared to α-AAA-B (3.12 ± 0.15 μM and 6.21 ± 0.17 μM respectively). Additive effect of amide analogs and γδ T cells showed significantly high leukemia cancer cell killing as compared to γδ T cells alone. A unique combinational therapy with γδ T cells and biologically active anti-cancer molecules (α-AAA-A/B), concomitantly may be a promising cancer therapy.Advanced stage cancers are aggressive and difficult to treat with mono-therapeutics, substantially decreasing patient survival rates. Hence, there is an urgent need to develop unique therapeutic approaches to treat cancer with superior potency and efficacy. This study investigates a new approach to develop a potent combinational therapy to treat advanced stage leukemia. Biologically active α-amino amide analogs (RS)-N-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)-N-phenylpropiolamide (α-AAA-A) and (RS)-N-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)-N-phenylbut2-enamide (α-AAA-B) were synthesized using linear Ugi multicomponent reaction. Cytotoxicities and IC Cancer is a disease characterized by uncontrolled growth of cells, causing mortality worldwide. In 2018, it was estimated that cancer was the second leading cause of death worldwide and was responsible for 9.6 million deaths ]. AccordCancer treatment options may include the use of various techniques ranging from surgery, radiation, medications and/or other therapies to cure, shrink or stop the progression of a cancer. Monotherapeutic treatments have limitations when it comes to advanced stage cancers, due to disease progression which makes the disease more complex . ScientiSome important molecules, such as Brentuximab Vedotin, Gemtuzumab Ozogamicin, Ado-trastuzumab emtansine, polatuzumab vedotin-piiq, and inotuzumab ozogamicin, are combined with monoclonal antibodies specific to surface antigens present on particular tumor cells and are used as combinational-targeted cancer therapies . There ain vitro), which favor these cells as an anticancer therapy option  work differently in immunotherapies for cancer. γδ T cells are one of the unconventional T cells, which can be distinguished from αβ T cells (major T cell subset). These cells express Vγ9Vδ2-TCR on their cell surface. Other than tumor killing, γδ T cells have numerous important functions in immunity, including cytokine production and mobilization of other types of immune cells  (RS)-N-(2-(Cyclohexylamino)-2-oxo-1-phenylethyl)-N-phenylpropiolamide (α-AAA-A) and (RS)-N-(2-(Cyclohexylamino)-2-oxo-1-phenylethyl)-N-phenylbut2-enamide (α-AAA-B), as anticancer agents and investigate their anti-proliferative or anti-metastatic activities alone or in combination with γδ T cells against leukemia cancer cell lines HL-60 and K562.™, 0.9% saline (NaCl), millipore filter (0.22 μM), fetus calf serum (FCS), L-glutamine , pen/strep , MEM sodium pyruvate (100 mM), non-essential amino acids . IL-2 (100 IU/ml) were from GIBCO Life Technologies, (USA). Human recombinant interleukin-2 and interleukin-15 were from Novartis (Switzerland) and Miltenyi Biotec (Germany), respectively. Mouse monoclonal antibodies specific for CD3 (UCHT1), TCR-Vγ9 (Immu360) were from Beckman Coulter (USA). Fixable aqua dead cell stain kit was from Invitrogen-Life Technologies (USA). Human IgG, methanol, aniline, benzaldehyde, propiolic acid, cyclohexyl isocyanide, dimethyl sulfoxide (DMSO) and 3--2,5-diphenyltratrazolium bromide were purchased from Sigma Aldrich (USA). Zoledronic acid injection (4 mg) were purchased from Cipla, (India). HL-60 and K562 cell lines were received from ATCC (USA).RPMI-1640 medium, lymphoprepBiologically active α-amino amide analogs (RS)-N-(2-(Cyclohexylamino)-2-oxo-1-phenylethyl)-N-phenylpropiolamide (α-AAA-A) and (RS)-N-(2-(Cyclohexylamino)-2-oxo-1-phenylethyl)-N-phenylbut2-enamide (α-AAA-B) were synthesized using linear Ugi multicomponent reaction (batch reaction with methanol) as published previously . BrieflyFor the synthesis of α-AAA-B, similar linear Ugi multicomponent reaction was used. A solution was prepared using methanol (5 ml), aniline  and benzaldehyde . This solution was stirred at 25°C for half an hour. Then 2-butyric acid  was added to this solution, followed by the addition of cyclohexyl isocyanide . This reaction mixture was stirred further at 25°C for 24 h till a precipitate formed, which was washed with diethyl ether and dried to obtain a white solid (0.21 gm).2.HL-60 and K562 were grown in complete RPMI 1640 medium which included nonessential amino acids and was supplemented with 10% FCS, 1 mM sodium pyruvate and 2 mM L-glutamate. All cells were grown at 37°C in 95% air with the addition of 5% CO5 cells/mL cancer cells (HL-60 or K562) in complete RPMI medium, with or without the addition of amide analogs (α-AAA-A or α-AAA-B), was followed by incubation for different durations (4–24 h) at 5% CO2 and 37°C. Thereafter, the cells were treated with 100 μl of MTT (5 mg/ml). Four hours later, the entire medium, including MTT solution, was aspirated from the wells. The remaining formazan crystals were dissolved in DMSO (50 μl) and absorbance was measured at 570 nm using a 96 well microplate reader. The cytotoxicity index was determined using the untreated cells as negative control. The percentage of cytotoxicity was calculated using the background-corrected absorbance as follows:The 3--2,5-diphenyltratrazolium bromide (MTT) assay has been done as described previously , 22 with6 cells/ml in 24-well culture plate and kept in CO2 (5%) incubator at 37°C for 2 weeks. The level of water in the incubator was checked often to prevent decrease in levels of medium. Cytokines IL-2 (100 IU/ml) and IL-15 (10 ng/ml) were added to the culture at day 3, 6, 8 and 11, and cells were split and fresh medium added. At day 14, the percent γδ T cells in culture were examined by flow cytometry. Fixable aqua dead cell stain kit was used to detect percent live γδ T cells. Total number of live cells was counted using trypan blue stain.γδ T cell lines were prepared as described in our previous research publication . BrieflyApproximately 100,000 to 500,000 PBMCs, before and after expansion, were plated in a total volume of up to 250 μl. These were centrifuged at 1100 rpm for 3 min at 4°C in a centrifuge. The cell pellets were then washed with 200 μl phosphate buffer saline (PBS). Three microliters of fixable aqua dead cell stain were added and incubated for 15 min in the dark, followed by the addition of 200 μl PBS containing 2% FCS (FACS buffer). After incubation, cells were centrifuged (1100 rpm for 3 mins) and the supernatant discarded. Cells were blocked with 100 μl of 1:1000 diluted human IgG for 15 min on ice. After incubation cells were centrifuged (1100 rpm for 3 mins) and pelleted. Again, the supernatant was discarded. Then cells were stained with 40 μl cocktails of antibodies & isotype by further incubating for 20 min on ice in the dark. Approximately 160 μl FACS buffer was added after incubation and then the mixture was centrifuged (1100 rpm for 3 min) again. Finally, cells were resuspended in 100 μl FACS buffer and were analyzed by flow cytometer .2 (5%) incubator at 37°C. Cancer cell-killing was determined by flow cytometry.Tumor cell-killing activity of expanded γδ T cell (14 days) was tested on the leukemia cancer cell lines (HL-60 and K562). Cancer cells were labeled with the membrane dye CellVue. Labeled cancer cells were incubated together with γδ T cells at increasing target cell (T) to effector (E) ratios  for 12 h in CO2 (5%) incubator at 37°C. Combinational cytotoxic effects of biologically active molecules (α-AAA-A and α-AAA-B) and expanded γδ T were analyzed for leukemia cancer cell lines (HL-60 and K562). α-AAA-A or α-AAA-B were added to the culture with different target (HL-60 or K652)/effector (γδ T cells) ratios. Cancer cells and γδ T cells alone served as controls (ctrl). For HL-60 cancer cell line, the concentrations used of α-AAA-A and α-AAA-B were 3.125 and 6.26 μM, respectively. For K-562 cancer cell line, concentrations used of α-AAA-A and α-AAA-B were 6.26 and 12.5 μM, respectively. Each assay is representative of three experiments.Combinational cancer cell killing was determined by flow cytometry. Cancer cells were labeled with the membrane dye CellVue. Labeled cancer cells were incubated together with γδ T cells at increasing target cell (T)/effector (E) ratios  for 12 h in COAll results were expressed as the mean ± SD. Multiple comparisons between data were done using software OriginPro 8.5 followed by Student’s t test. The p value for significance was set at < 0.05.Most of the drugs used in the treatment of leukemia cause severe toxicities and, hence, it is important to synthesize and test novel biologically active molecules which can potentially be used as anti-cancer agents with low toxicities. Two biologically active compounds Figure 1p < 0.001) were observed in percent cell inhibition of HL-60 cells between 1.56 and 3.125 μM concentrations after 12 and 24 h of incubations  when compared with concentrations 6.25 and 12.5 μM (Cancer cell line K562 cells showed high cytotoxicity at higher concentrations of 6.25 and 12.5 μM when incubated with α-AAA-A (p < 0.001) when compared with 12.5 μM  values were extrapolated from the concentration-response log10 graphs, using MTT assay. The IC50 values were calculated for HL-60  were stimulated using 1 µM zoledronate and were cultured in complete RPMI 1640 medium. Cytokines IL-2 (100 IU/ml) and IL-15 (10 ng/ml) were added to the culture according to the protocol using fresh medium. After fourteen days of culture, high yield (2279.2 ± 487) of pure γδ T (90.7 ± 4.6%) cells was recovered  in killing of both HL-60 and K562 cells at the target (T) and effector (E) ratios of 1:10, 1:25 and 1:50 as compared to 1:1 T:E ratio. However, there are also significant differences (P < 0.01) between both cell lines in percent cell killing on comparison of T:E ratios of 1:10 and 1:25. Highest tumor cell killing for HL-60 cells was evident at a T:E ratio of 1:25 (41%). Similarly, highest killing for K562 cells was achieved at a T:E ratio of 1:25 (33%). It is evident that lesser numbers of γδ T cells  are not able to successfully kill tumor cells. Also, γδ T cells kill HL-60 cells more effectively as compared with K562 cells. γδ T cells as well as both leukemia cancer cell lines (HL-60 and K562), when present alone, did not show substantial cell killing after 12 h of incubation under similar culture conditions.Freshly expanded γδ T cells showed cytotoxicity toward HL-60 (in vitro for their cytotoxic effects on leukemia cancer cell lines (HL-60 and K562) using flow cytometer. Significantly high percent of cancer cell killing was observed when these cell lines were co-cultured with molecule (α-AAA-A or α-AAA-B) and γδ T cells together. Highest percent of killings of HL-60 cells (72%)  and expanded γδ T cells were tested The control experiments were designed for co-culture of expanded γδ T cells with both molecules (α-AAA-A and α-AAA-B) at various concentrations to analyze the effect of α-amino amide analogs on 14 days expanded cells. Results showed no substantial cytotoxic effects (≤ 4%) on these cells  at a target to effector ratio of 1:25. We did not further elucidate the differences in killing of two different cell lines. In our previous study, we have also shown the cytotoxic effects of these cells against another chronic myeloid leukemia cell line, i.e., KBM7 . However, direct blocking assays were not carried out in this study and would be included in future studies to elucidate the mechanism underlying cytotoxicity.in vitro studies also showed that γδ T cells kill breast cancer cell lines MDA-MB231, MCF-7, and T47D  .in vitro conditions and both can be generated in large numbers or amounts. Therefore, both biologically active α-amino amide analogs and γδ T cells are ideal candidates for their use in cancer therapy as combinational therapeutics.Pathways for cancer cells are difficult to understand, often due to the involvement of multiple complex molecules. Use of single drug or vaccine poses limitations in countering the complex pathogenesis of cancer. In light of these difficulties, combinational therapies present a unique approach and may provide effective outcomes for many different cancers , 24. ImpMany studies conducted so far have successfully demonstrated the use of immune cells  with small biological molecules  which are immune check point inhibitors . SimilarToxicity and increase of multidrug resistance in cancer patients is a major constraint in chemotherapy . Hence, in vitro conditions. Thus, biologically active molecule (α-AAA-A and α-AAA-B) and γδ T cells are potential agents for combinational therapy for leukemia. In the future, anticancer molecules may be engineered to perform dual function; first to exhibit cancer cell killing and second to activate in vivo γδ T cells.High yields of novel biologically active molecules (α-AAA-A and α-AAA-B) were achieved with simple reactions, minimum efforts and without any purification. These molecules exhibited cytotoxic activities against leukemia cancer cell lines and remain stable at room temperature. Biologically active molecules can reach a wide spectrum of molecular targets, including intracellular targets or those present deep in the tumor micro-environment. Human γδ T cells exhibit tumor killing activity. The combination of α-AAA-A or α-AAA-B with γδ T cells effectively killed HL-60 and K562 cancer cells in The original contributions presented in the study are included in the article/This study has been reviewed and approved by the Research Ethics Committee (REC) at the University of Hail dated: 27/11/2020 and approved by university president letter number Nr. 20455/5/42 dated 16/04/1442 H.AO, SS, and MK designed the study. AO, SS, WK, SK, and MK performed the study. AO, SS, MK, SA, EA, and AA wrote the manuscript. All authors contributed to the article and approved the submitted version.This research has been funded by Research Deanship of University of Ha’il, Saudi Arabia through project number RG-191332.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.


H-1,2,3-dithiazol-5-one and two polymorphs of 4-chloro-5H-1,2,3-dithiazole-5-thione were analyzed and compared to structural data of similar systems. These five-membered S,N-rich heterocycles are planar with considerable bond localization. All three structures demonstrate tight solid-state packing without voids which is attributed to a rich network of short intermolecular electrostatic contacts. These include Sδ+…Nδ−, Sδ+…Oδ−, Sδ+…Clδ− and Sδ+…Sδ− interactions that are well within the sum of their van der Waals radii (∑VDW). B3LYP, BLYP, M06, mPW1PW, PBE and MP2 were employed to calculate their intramolecular geometrical parameters, the Fukui condensed functions to probe their reactivity, the bond order, Bird Index and NICS(1) to establish their aromaticity.The crystal structure and solid-state packing of 4-chloro-5 These interactions (1  and 3.5316(7) Å  well within the ∑ractions  are longtions . The location of the oxygen atom is, therefore, ideal to create a bifurcated set of Cδ+…Oδ− contacts (VDW   and 3.066(2) Å . Each carbon atom of the C–C bond has a significant partial positive charge stemming from the polarization of the C–Cl and C=O bonds due to the difference in electronegativity (Ccontacts . These C(major)] .2b is concluded with a S…S contact 3.4771(7) Å  between two endocyclic S atoms from neighboring molecules. This represents a short contact due to the proximity of the S atoms. Both atoms are expected to have a partial positive charge Sδ+ but since the electron cloud around S is highly polarizable this could also be an electrostatic interaction.The network of intermolecular contacts for dithiazolone 2b stack along the c-axis , 3.63 Å (major)].The δ+…Nδ−) and therefore electrostatically favored.The crystal packing of S,N-rich heterocycles is dominated by the presence of short S…N intermolecular contacts. S-N bonds are strongly polar (SVDW (3.20 Å). Neighboring molecules form centrosymmetric pairs linked by short S…N interactions of 3.035(2) Å. No S…N contacts appear in the reported crystal structures of the benzoyldithiazolone 3 and dithiazolethiones 4 and 5  between two endocyclic S atoms from neighboring molecules. A similar interaction appears in the crystal packing of dithiazolone 2b.There are two S…Cl intermolecular interactions of 3.427(1) Å  and 3.445(1) Å  well within the ∑VDW . This is a near triangular interaction of the same covalently bound Cl to two S atoms of neighboring dithiazoles . The dis packing . Instead packing . The thing rings . These Sα2c– form zig-zag chains along the a-axis  and 3.427(1) Å, respectively. Two chains, linked by S…Cl contact of 3.445(1) Å, run parallel across c-axis to form a ribbon. Neighboring ribbons run antiparallel to each other and are connected by S…S and S…N contacts of 3.448(2) and 3.251(4) Å, respectively, to form a 2D sheet , 4.06 Å (major)]. While this type of interaction is also seen in a2c–, the highly polarizable nature of sulfur allows for the formation of interactions between the exocyclic thione S atom and the more electronegative N and Cl atoms. This type of interaction originates from the positive σ hole of the S atom. In the C=S bond, some of the electronic charge of the S atom is polarized toward the bond region, leading to a redistribution of electronic density from its outer region  on its equatorial sides  and well within the ∑VDW . The exocyclic thione S atom also formed an interaction with a Cl atom. This S…Cl contact is in the range of 3.315(4)–3.418(4) Å . The set of close intermolecular interactions in β2c– is completed with a Cl…Cl contact of 3.337(4)–3.342(4) Å  not present in the crystal packing of a2c– or compounds 2b, 3–5. This interaction might be an outcome of the proximity of the dithiazolethiones.The thione S atoms in ole ring . These Sal sides . Therefohione 2c  where reac-diagonal and while well separate from each other .2b. The difference between the experimental bond angles and computed bond angles are mostly < 1° except for the S2–N3–C4 bond angle. This latter bond angle ranges from −1.6% error for B3LYP and M06 to −0.2% for MP2. On average the differences of either the DFT or MP2 are < 1% , nucleophilic (f−) and radicalary attack (f0) Fukui functions based on NBO population analysis , wR2 = 0.0663 , and S = 1.112 with the largest difference peak and hole of 0.264 and −0.381 e−3, respectively.Crystal refinement data (CCDC 2106260): C2ClNS3, M = 169.66, Orthorhombic, space group P b c a, a = 11.0262(5) Å, b = 7.2698(3) Å, c = 13.3111(8) Å, α = β = γ = 90°, V = 1067.00(9)Å3, Z = 8, T = 100(2) K, ρcalcd = 2.112 g cm−3, 2θmax = 67. Refinement of 64 parameters on 953 independent reflections out of 5321 measured reflections (Rint = 0.1001) led to R1 = 0.0680 [I > 2σ(I)], wR2 = 0.1993 , and S = 1.077 with the largest difference peak and hole of 0.836 and −1.097 e−3, respectively.Crystal refinement data (CCDC 2106261): C2ClNS3, M = 169.66, Triclinic, space group P-1, a = 7.113(5) Å, b = 13.323(5) Å, c = 18.343(5) Å, α = 76.886(5)°, β = 82.914(5)°, γ = 76.429(5)°, V = 1641.2(14) Å3, Z = 12, T = 100(2) K, ρcalcd = 2.060 g cm−3, 2θmax = 25. Refinement of 379 parameters on 5782 independent reflections out of 10938 measured reflections (Rint = 0.0911) led to R1 = 0.0845 [I > 2σ(I)], wR2 = 0.2120 , and S = 0.981 with the largest difference peak and hole of 0.228 and −0.806 e−3, respectively.Crystal refinement data (CCDC 2106262): Cf−), electrophilic (f+) and radical attack (f0) characterize the electron reorganization that results in site electrophilic or nucleophilic activation/deactivation [Our crystallographic data provided the initial geometry for the pair of structures. Each structure was energy optimized by density functional theory. The combinations of exchange and correlation functionals used in this work include B3LYP ,60,61,62tivation . Calculativation  populatitivation .