Management of bladder cancer (BLCA) has not changed significantly in the past few decades, with platinum agent chemotherapy being used in most cases. Chemotherapy reduces tumor recurrence after resection, but debilitating toxicities render a large percentage of patients ineligible. Recently approved immunotherapy can improve outcomes in only a third of metastatic BLCA patients. Therefore, more options for therapy are needed. In this study, we explored the efficacy of PARP inhibitors (PARPi) as single agents or as combinations with platinum therapy.We treated BLCA cells with PARPi  alone or as the combination of cisplatin with PARPi. We then measured their survival, proliferation, apoptosis, as well as their ability to form colonies. BLCA xenografts in male SCID mice were treated similarly, followed by the assessment of their growth, proliferation, and apoptosis.In vivo, the combinations of cisplatin with niraparib, rucaparib, or talazoparib reduced BLCA xenograft growth significantly.PARPi niraparib and talazoparib were effective in reducing BLCA cell survival as single agents. Combinations of Cisplatin with talazoparib and niraparib effectively reduced the survival of BLCA cells, while veliparib was not effective even at high concentrations. We provide evidence that PARPi can be effective against BLCA as single agents or as combinatorial therapy with cisplatin.The online version contains supplementary material available at 10.1186/s12885-022-09376-9. Bladder cancer (BLCA) is the sixth most common cancer in the US and the ninth most common cancer worldwide , 2. WhilThe previous decades have not seen significant changes in the management of BLCA, with the combination of methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) or gemcitabine with cisplatin being used as the systemic therapy of choice since 1985 –9. AdjuvGenomic DNA that is damaged due to free radicals, mutagens, or carcinogens is repaired by the Poly (ADP-Ribose) Polymerase family proteins. Seventeen members of the PARP family are known, with PARP1 accounting for most DNA damage repair (DDR) events in malignant and non-malignant cells. PARP1 binds to single strand breaks (SSB) and recruits a cascade of DDR proteins. Recruitment of these proteins results in PARP dissociation from DNA and SSB repair . In cellPARPi were thought to be of little value in cancers such as prostate and bladder owing to the fact that inheritable BRCA mutations occur with low frequency in such cancers. Nevertheless, recent reports indicate that the usefulness of PARPi can encompass other perturbations in the HR pathway –22. ManyFew earlier studies offer definitive conclusions verifying the efficacy of PARPi in BLCA. Given that HR gene mutations amplify the effects of DNA damage caused by ionizing radiation or platinum drugs, PARPi may also exhibit efficacy as adjunctive therapy with cisplatin or carboplatin. The current study was aimed to compare 5 commercially available PARPi in BLCA cells and test their efficacy in combination with cisplatin. Understanding the mechanisms and the association of HR repair defects with sensitivity to PARPi can signal a breakthrough in BLCA therapy. Established BLCA cell lines were chosen for the study based on observations that BLCA cell lines replicate many genetic aberrations associated with BLCA tumor development . Our reshttps://cancer.sanger.ac.uk/cosmic, https://cancergenome.nih.gov/, and http://www.cbioportal.org/). We sought to determine the correlation between mutations in DDR pathway genes and BLCA patient survival. The list of DDR genes for which we sought to determine mutational status is from [Publicly available databases containing mutational data for HR pathway genes were queried using COSMIC, The Cancer Genome Atlas, and cBioPortal . Tubulin antibodies were obtained from Thermo Fisher Scientific, Waltham, MA. Cleaved and whole caspases 3, 7, and 9 antibodies were obtained from Cell Signaling Technology, Danvers, MA. Ki-67 antibodies were obtained from Neomarker, Fremont, CA. The PARP inhibitors, olaparib, niraparib, rucaparib, veliparib, and talazoparib, were obtained from MedChem Express, Monmouth Junction, NJ. Cisplatin was from Sigma Aldrich, St. Louis, MO. Other reagents were supplied by local suppliers such as Fisher Scientific and VWR International.UM-UC-3, T-24 (human bladder cancer cell lines), and SV-HUC-1  were obtained from the American Type Culture Collection  and were cultured in EMEM, McCoy’s 5a, or F12K media respectively, supplemented with 10% FBS and penicillin/streptomycin. Cells were used within half a year after being received from ATCC or after thawing from cryopreservation. Short Tandem Repeat (STR) profiling is used by the ATCC for cell line authentication. The cell lines in culture were routinely tested for mycoplasma contamination every two months using the MycoFluorSV-HUC-1, UM-UC-3, or T-24 cells were seeded at 1000 cells/well in 96-well plates and treated with PARPi or their combinations with cisplatin as shown in the respective figures. A Coulter cell counter  was used to determine cell viability.SV-HUC-1, UM-UC-3, or T-24 cells were seeded at 1000 cells/well in 96-well plates and treated with PARPi or their combinations with cisplatin for 3 days. The CellTiter 96® Aqueous One Solution Cell Proliferation kit  was used according to manufacturer instructions to assess cell proliferation.High salt buffer containing 50 mM Hepes pH 7.9, 250 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, 1 mM Na Vanadate, 1 mM NaF, and protease inhibitors (Roche) was used to lyse cells as described earlier . The CoooC for 10-14 days. The colonies were stained with 0.5% Crystal Violet in buffered formalin and colony numbers were counted using ImageJ.Anchorage-dependent clonogenicity was assayed as described in earlier studies . SV-HUC-oC in sterile cages. We injected 2 million UM-UC-3 cells sub-cutaneously in a 1:1 (v/v) ratio with matrigel into both flanks of mice and monitored tumor growth. After average tumor volumes reached 0.1 cm3 approximately, we divided the mice randomly into 10 groups (n = 5/group). The treatment groups were: 1) 0.5% Methocel A4M as vehicle control, 2) niraprib at 10 mg/kg, 3) olaparib at 25 mg/kg, 4) rucaparib at 50 mg/kg, 5) talazoparib at 0.5 mg/kg delivered via daily oral gavage, 6) cisplatin at 0.5 mg/kg delivered intra-peritoneally once every two days, 7) cisplatin+niraparib, 8) cisplatin+olaparib, 9) cisplatin+rucaparib, or 10) cisplatin+talazoparib. Mice were treated for three weeks and growth of the tumors and mouse weights were measured using digital calipers or a balance every other day. Tumor growth was used as the outcome measure. When the control tumors reached an average of 1500 mm3, mice in all groups were euthanized with carbon dioxide followed by cervical dislocation. No animals were excluded from any analyses. Tumor inhibition was calculated as percentage tumor growth inhibition compared with vehicle control. We harvested the xenograft tissues and analyzed the expression of ki-67, cleaved caspases 3, 7, and 9 with immunohistochemistry. All experiments with animals were governed by the Institutional Animal Care and Use Committee of the University of Toledo (IACUC protocol # 108804) and were performed in line with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Mouse xenograft models are commonly used to assess the efficacy of therapeutic strategies. A total of 60 male 4–5-week-old SCID mice  with an average weight of 20 g were used in this study. Mice were allowed to acclimate for 7 days after receipt from the vendor and were housed at 22.5 ± 0.5Immunohistochemistry was performed as described earlier . Tumor tResults are reported as means ± SD. One-way ANOVA was used for the comparison of multiple groups with alpha set at 0.05. A P value cut-off ≤0.05 was established to indicate significance. All data were analyzed using the Microsoft Excel Data Analysis Toolpak for Windows 10 .We used GDC Data Portal, COSMIC, and cBioPortal to analyze mutation rates in DNA repair genes  in BLCA 50 of cisplatin as well as the PARPi  in UM-UC-3 cells by treating with 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100, and 1000 μM concentrations for 72 h. The AAT Bioquest  online tool was used to calculate IC50 values. The IC50 curves and the calculated IC50 values for each of these agents are summarized in Fig. 50 values (μM) were as follows: Niraparib (8.6093); olaparib (8.2312); rucaparib (15.5063); talazoparib (1.0989); veliparib (39.4209); and cisplatin (3.163).We determined the ICNext, to determine working concentrations for each of the PARPi, we treated UM-UC-3 and T-24 as well as SV-HUC-1 cells with 5, 10, or 20 μM of olaparib, niraparib, veliparib, or rucaparib or 0.5, 1, or 2 μM of talazoparib for 3 days. As demonstrated in Fig. 50 concentrations of PARPi for 5 days. As shown in Fig. p = 0.034), 61.43 ± 13.24 (p = 0.011), and 47.71 ± 9.58 (p = 0.0012), respectively, compared with their respective DMSO-treated controls. The percentages of proliferation in olaparib-treated SV-HUC-1, UM-UC-3, and T-24 cells were 84.67 ± 3.10 (p = 0.041), 70.11 ± 6.36 (p = 0.035), and 76.39 ± 1.84 (p = 0.022), respectively, compared with their respective DMSO-treated controls. The percentages of proliferation in rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 cells were 90.84 ± 3.41 (p = 0.07), 79.78 ± 0.85 (p = 0.039), and 83.03 ± 4.51 (p = 0.046), respectively. The percentages of proliferation in talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 cells were 94.22 ± 5.18 (p = 0.43), 75.54 ± 0.66 (p = 0.036), and 81.61 ± 2.42 (p = 0.047), respectively, compared with their respective DMSO-treated controls. These results indicated that PARPi suppressed the proliferation of UM-UC-3 and T-24 more significantly compared with that of SV-HUC-1 cells, suggesting that PARPi can be used as potential therapeutic agents against BLCA.We treated BLCA cell lines UM-UC-3 and T-24 and the normal urothelial cells SV-HUC-1 with sub-IC50 concentrations of PARPi for 72 h and performed clonogenic assays as described earlier [p = 0.036), 56.44 ± 5.62 (p = 0.022), and 74.43 ± 8.30 (p = 0.013), respectively, compared with their DMSO-treated controls. The percentages of colonies formed in olaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 48.84 ± 4.36 (p = 0.0021), 76.99 ± 2.12 (p = 0.037), and 85.52 ± 5.75 (p = 0.047), respectively, compared with their DMSO-treated controls. The percentages of colonies formed in rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 70.76 ± 2.66 (p = 0.033), 87.42 ± 7.53 (p = 0.048), and 94.73 ± 4.4 (p = 0.71), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 45.76 ± 9.81 (p = 0.0019), 56.44 ± 2.81 (p = 0.0031), and 68.23 ± 0.97 (p = 0.002), respectively, compared with their respective DMSO-treated controls. The results showed that PARPi inhibited the clonogenic ability of BLCA cells and normal urothelial cells significantly , 96.09 ± 4.8 (p = 0.42), and 98.85 ± 1.66 (p = 0.31), respectively, compared with their DMSO-treated controls. The percentages of cells surviving in niraparib-treated vs. CP+Niraparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 18.44 ± 7.56 vs. 11.85 ± 1.74 (p = 2.663e-8), 55.34 ± 3.10 vs. 22.19 ± 7.56 (p = 1.57e-6), and 24.33 ± 3.53 vs. 23.44 ± 8.49 (p = 0.00059), respectively, compared with their respective DMSO-treated controls. The percentages of cells surviving in olaparib-treated vs. CP+Olaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 56.14 ± 6.08 vs. 40.03 ± 3.54 (p = 3.69e-4), 36.75 ± 6.22 vs. 20.84 ± 1.08 (p = 1.22e-5), and 52.83 ± 1.86 vs. 31.69 ± 4.21 (p = 3.19e-3), respectively, compared with their respective DMSO-treated controls. The percentages of cells surviving in rucaparib-treated vs. CP+Rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 42.53 ± 7.03 vs. 31.04 ± 2.57 (p = 4.17e-4), 62.60 ± 4.29 vs. 34.29 ± 1.32 (p = 3.81e-4), and 54.20 ± 2.93 vs. 31.05 ± 0.86 (p = 6.43e-4), respectively, compared with their respective DMSO-treated controls. The percentages of cells surviving in talazoparib-treated vs. CP+Talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 35.35 ± 4.38 vs. 22.22 ± 5.20 (p = 4.21e-6), 47.01 ± 0.69 vs. 27.95 ± 4.16 (p = 0.0014), and 36.63 ± 1.41 vs. 30.48 ± 2.67 (p = 2.19e-3), respectively, compared with their respective DMSO-treated controls. The percentages of cell proliferation in niraparib-treated vs. CP+Niraparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 77.75 ± 8.63 vs. 70.74 ± 0.32 (p = 0.049), 61.43 ± 13. 24 vs. 37.49 ± 1.30 (p = 0.031), and 47.71 ± 9.58 vs. 33.97 ± 3.06 (p = 0.015), respectively, compared with their respective DMSO-treated controls. The percentages of cell proliferation in olaparib-treated vs. CP+Olaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 84.67 ± 3.10 vs. 74.14 ± 1.89 (p = 0.06), 70.11 ± 6.36 vs. 36.39 ± 3.83 (p = 0.037), and 76.39 ± 1.84 vs. 45.69 ± 3.24 (p = 0.022), respectively, compared with their respective DMSO-treated controls. The percentages of cell proliferation in rucaparib-treated vs. CP+Rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 90.84 ± 3.41 vs. 75.45 ± 11.12 (p = 0.072), 79.78 ± 0.85 vs. 41.35 ± 3.77 (p = 0.043), and 83.03 ± 4.51 vs. 42.32 ± 9.77 (p = 0.044), respectively, compared with their respective DMSO-treated controls. The percentages of cell proliferation in talazoparib-treated vs. CP+Talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 94.22 ± 5.18 vs. 76.56 ± 14.34 (p = 0.069), 75.54 ± 0.66 vs. 44.24 ± 8.88 (p = 0.016), and 81.61 ± 2.42 vs. 43.22 ± 1.6 (p = 0.0449), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in niraparib-treated vs. CP+Niraparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 72.30 ± 2.66 vs. 57.30 ± 1.33 (p = 0.044), 56.44 ± 5.62 vs. 45.09 ± 1.59 (p = 0.021), and 74.43 ± 8.3 vs. 34.21 ± 5.23 (p = 0.031), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in olaparib-treated vs. CP+Olaparib-treated SV-HUC-1, UM-UC3, and T-24 groups were 48.84 ± 4.36 vs. 28.07 ± 2.90 (p = 0.002), 76.99 ± 2.12 vs. 62.88 ± 5.62 (p = 0.023), and 85.52 ± 5.75 vs. 63.90 ± 5.86 (p = 0.049), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in rucaparib-treated vs. CP+Rucaparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 70.76 ± 2.66 vs. 70 ± 5.45 (p = 0.073), 87.42 ± 7.53 vs. 70.55 ± 6.46 (p = 0.066), and 94.73 ± 4.40 vs. 77.25 ± 3.94 (p = 0.048), respectively, compared with their respective DMSO-treated controls. The percentages of colonies formed in talazoparib-treated vs. CP+Talazoparib-treated SV-HUC-1, UM-UC-3, and T-24 groups were 45.76 ± 9.81 vs. 28.84 ± 5.02 (p = 2.47e-5), 56.44 ± 2.81 vs. 37.42 ± 4.61 (p = 1.22e-3), and 68.23 ± 0.97 vs. 43.98 ± 6.50 (p = 0.0031), respectively, compared with their respective DMSO-treated controls. The combinations of PARPi with sub-IC50 concentrations of cisplatin inhibited cell survival , cisplatin, or cisplatin+different PARPi [3. Niraparib  and rucaparib  reduced tumor growth to a similar extent singly as well as in combination with cisplatin   (Fig. To establish that PARPi can synergize with cisplatin treatment nt PARPi . ToxicitCisplatin-based chemotherapy has been used as the first line therapy for locally advanced BLCA for decades . HoweverPARP inhibition relies on the presence of mutations or alterations in DNA damage genes such as those involved in HR. In-silico analyses revealed that ~30% of BLCA harbor mutations in HR genes, indicating that PARP inhibition can be used in BLCA. Earlier studies showed that PARP inhibition combined with cisplatin significantly increased lifespan and restored nerve conduction velocity in animal models . PARP inThe mechanism of action of PARPi includes: catalytic inhibition of PARP or “PARP trapping”, in which PARP is trapped at sites of DNA damage leading to prevention of repair and cytotoxicity. It has been postulated that PARP trapping activity may be indicative of higher efficacy in a PARPi . The relDefects in DDR can predict response to cisplatin-based chemotherapy in muscle-invasive BLCA . AlteratWe demonstrated that PARP inhibitors can not only be effective against BLCA as single agents but can also be used in combinations to improve the efficacy of first-line therapeutic regimen. The current study is one of the first studies to compare all 5 commercially available PARP inhibitors and examine their relative outcomes in combination with cisplatin in BLCA. Given that PARPi are already undergoing clinical evaluation in urothelial bladder cancer , these pAdditional file 1.Additional file 2.Additional file 3.


T, was isolated from seawater of the West Pacific Ocean. Cells were non-motile and rod-shaped, with carotenoid-type pigments. Strain A3-108T grew at pH 6.0–8.5 (optimum 6.5) and 15–40 °C (optimum 28 °C), in the presence of 0.5–10% (w/v) NaCl (optimum 1.0%). It possessed the ability to produce H2S. Based on the 16S rRNA gene analysis, strain A3-108T exhibited highest similarity with Aureisphaera salina A6D-50T (90.6%). Phylogenetic analysis shown that strain A3-108T affiliated with members of the family Flavobacteriaceae and represented an independent lineage. The principal fatty acids were iso-C15:0, iso-C17:0 3-OH, iso-C15:1 G, and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The sole isoprenoid quinone was MK-6. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified aminolipid and one unidentified lipid. The ANIb, in silico DDH and AAI values among the genomes of strain A3-108T and three reference strains were 67.3–71.1%, 18.7–22.1%, and 58.8–71.4%, respectively. The G + C content was 41.0%. Distinctness of the phylogenetic position as well as differentiating chemotaxonomic and other phenotypic traits revealed that strain A3-108T represented a novel genus and species of the family Flavobacteriaceae, for which the name Luteirhabdus pelagi gen. nov., sp. nov. is proposed .A Gram-stain-negative, aerobic, and yellow-pigmented bacterium, designated A3-108The online version contains supplementary material available at 10.1007/s00203-021-02557-3. Flavobacteriaceae, belonging to the class Flavobacteriia, the phylum Bacteroidetes, was first proposed by Jooste , 0.1 g yeast extract (BD Difco), 20 g agar (BD Difco), pH 7.2–7.4) and purified by repeated restreaking. The purity was confirmed by the uniformity of cell morphology. Unless otherwise stated, strain A3-108T was routinely cultured in marine broth 2216  or on marine agar 2216  at 30 °C and maintained at − 80 °C with 30% (v/v) glycerol. Galbibacter mesophilus CGMCC 1.15663T and Marixanthomonas ophiurae JCM 14121T, were obtained from the CGMCC  and JCM (Japan Collection of Microorganisms), respectively. An additional reference strain Marinirhabdus gelatinilytica NH83T was obtained from our laboratory , was collected by a rosette sampler connected with CTD system  in 2018. Aboard the ship, the seawater sample was subjected to the culture process immediately. Approximately 100 µL seawater samples were diluted using serial dilution technique and added to different media. The strain A3-108High-quality genomic DNA was extracted by Nucleic Acid Purification kit (Dongsheng Biotech). The 16S rRNA gene was amplified by the universal primers 27F/1492R . The PCR thermal cycling conditions were as follows: 30 cycles of 98 °C for 10 s, 55 °C for 10 s, and 72 °C for 30 s. The PCR products were purified and sequenced by Sanger sequencing to obtain the almost complete 16S rRNA gene sequence.T and Galbibacter mesophilus CGMCC 1.15663T were sequenced by Solexa paired-end sequencing technology with the Illumina NovaSeq 6000 PE150 platform . One paired-end library was constructed with insert size of 350 bp. The sequencing generated approx. 1 Gb clean data (approx. 500-fold genome coverage). De novo assembly of the reads was carried out using SOAPdenovo (version 2.0.1)    of strain A3-108 Seemann . Ortholo Seemann . Single- Seemann . Aligned Seemann  and conc Seemann  and the  Seemann .http://jspecies.ribohost.com/jspeciesws/), Genome-to-Genome Distance Calculator  (https://ggdc.dsmz.de/home.php) and AAI calculator (http://enve-omics.ce.gatech.edu/aai/), respectively  values, the DNA–DNA hybridization (DDH) values and the average amino acid identity (AAI) values were calculated using JSpeciesWS , and cell densities were measured every 2 h incubation via measuring OD600 in a UV/Visible Spectrophotometer . The doubling time and the specific growth rate were calculated by formulas:Cell morphology, ultrastructure, size, and the presence of flagellum were observed by transmission electron micrographs . Gram reaction was determined by the Gram-Stain method (Brown and Hopps td/h) = ln2/k; specific growth rate (μ/h−1) = 1/td;doubling time (k represents relative growth rate (slope of the curve) (Monod e) Monod .Flexirubin-type pigments were detected by a bathochromic shift test , sodium sulfate , and cysteine  and the hydrolysis abilities of starch , DNA ), L-tyrosine , esculin , CM-cellulose , and Tween 40 , 60 , and 80  were determined as previously described . Fatty acids were extracted by saponification, methylation, and extraction as described previously (Sasser The cellular fatty acids of the strain A3-108y Sasser . The celT was cultivated in MB at 30 °C for 3 days to obtain cell biomass. Respiratory quinones were extracted from cells (around 200 mg) with chloroform/methanol  and analyzed by LC–MS (Agilent)  , followed by Galbibacter mesophilus Mok-17T (90.5%), Marinirhabdus gelatinilytica NH83T (90.4%), Aureisphaera galaxeae 04OKA003-7T (90.4%) and Aequorivita aestuarii JC2436T (90.2%).The almost complete 16S rRNA gene sequence of strain A3-108T fall into the family Flavobacteriaceae and formed a separated branch apart from other genera of the family with high bootstrap values  demonstrated that strain A3-108T affiliated with the family Flavobacteriaceae and clustered with Marixanthomonas ophiurae KMM 3046T . The ANI values were far below the species threshold of 94–96% (Richter and Rosselló-Móra T and the reference strains (Supplementary Table S2), which were below the species cutoff 95–96% (Konstantinidis and Tiedje T and the reference strains were 67.7–71.5% (Supplementary Table S2). The ANI, in silico DDH and AAI values indicted a low taxonomic relatedness between strain A3-108T and the reference strains of the family Flavobacteriaceae.Based on the bioinformatic tool CheckM, the genome completeness of strain A3-108T was Gram-stain-negative, aerobic, non-motile, and rod-shaped with 0.5–0.8 μm in width and 1.6–3.9 μm in length . No flagellum was observed. Colonies were yellow, circular, convex, opaque, smooth, and 1–2 mm in diameter after 3 days of incubation at 30 °C on MA. The growth range of pH, temperature and NaCl concentrations were pH 6.0–8.5, 15–40 °C, and 0.5–10% (w/v) on MB. The growth of strain A3-108T contained three phases, including the lag phase (0–16 h), exponential phase (16–40 h), and stationary phase (exceed 40 h) at 30 °C on MB. The doubling time and the specific growth rate were 6.2 h and 0.16 h−1, respectively . Strain A3-108T was positive for oxidase, arginine dihydrolase, nitrite reduction, and H2S production. Carotenoid-type pigments were present but flexirubin-type pigments were not . Additional phenotypic properties are given in the species description, Tables Strain A3-108T was menaquinone-6 (MK-6). Strain A3-108T possessed phosphatidylethanolamine (PE), one unidentified aminophospholipid (APL), one unidentified aminolipid (AL) and one unidentified lipid (L1) as major polar lipids. In addition, two unidentified aminoglycolipids (AGL1-2), one unidentified glycolipid (GL), and two unidentified lipids (L2-3) were present as moderate or minor lipids . The major fatty acids of strain A3-108T contained iso-C15:0 (22.4%), iso-C17:0 3-OH (17.2%), iso-C15:1 G (15.7%), and summed feature 3 (C16:1ω7c and/or C16:1ω6c) (10.3%) , while it was presented as moderate fatty acid in Marinirhabdus gelatinilytica NH83T, Galbibacter mesophilus CGMCC 1.15663T and Marixanthomonas ophiurae JCM 14121T (3.2–7.8%). In addition, the component anteiso-C15:1 A was only detected in the strain A3-108T. In addition, the fatty acid of strain A3-108T were different from the reference strains in the compositions and proportions , one of the major polar lipids, was not presented in the related strain Marixanthomonas ophiurae JCM 14121T. In addition, moderate polar lipids, including an unidentified glycolipid (GL) and two aminoglycolipids (AGL1-2), were presented in the strain A3-108T, while they were not detected in the reference strains. Besides, lysophosphatidylethanolamine (LPE) and sphingolipid (SL) were only detected in Marixanthomonas ophiurae JCM 14121T .2S production. The predominant menaquinone is MK-6. Major polar lipids are phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified aminolipid, and one unidentified lipid. The major cellular fatty acids are iso-C15:0, iso-C17:0 3-OH, iso-C15:1 G, and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The DNA G + C content of the type species is 41.0%. The genus belongs to the family Flavobacteriaceae, class Flavobacteriia, phylum Bacteroidetes. The type species is Luteirhabdus pelagi.Cells are Gram-stain-negative, strictly aerobic, non-motile and rod-shaped. No flagellum was observed. Carotenoid-type pigments are produced. Positive for oxidase, HLuteirhabdus pelagi (pe.la'gi. L. gen. n. pelagi of the open sea).+ ions for growth. Growth occurs in NaCl-free MB supplement with 0.5–10% (w/v) NaCl (optimum at 1.0%). The pH and temperature ranges for growth are pH 6.0–8.5 and 15–40 °C (optimum at pH 6.5 and 28 °C). Carotenoid-type pigments are produced but flexirubin-type pigments are not. No anaerobic growth occurs on MA supplemented with sodium nitrate, sodium sulfate, and sodium thiosulfate. Positive for oxidase, arginine dihydrolase, gelatin, and nitrite reduction. Negative for catalase, indole production, glucose fermentation, urease, and nitrate reduction. Negative for the degradation of Tweens 40, Tweens 60, L-Tryosine, starch, esculin, CM-cellulose, DNA, and β-galactosidase. H2S production occurs on MB supplemented with sodium thiosulfate, cysteine and sodium sulfate. Acid and alkaline phosphatase, α-chymotrypsin, cystine arylamidase, esterase (C4), esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase activities are present. Acid is not produced from citrate, D-cellobiose, D-fructose, D-galactose, D-glucose, D-maltose, D-mannose, D-salicin, D-trehalose, D-xylose, L-arabinose, L-malate, L-glutamic acid, and sucrose. The principal fatty acids are iso-C15:0, iso-C17:0 3-OH, iso-C15:1 G, and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The sole respiratory quinone is menaquinone-6 (MK-6). The major polar lipids are phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified aminolipid, and one unidentified lipid. In addition, moderate to minor amounts of two unidentified aminoglycolipids, one unidentified glycolipid, and two unidentified lipids are present. The DNA G + C content is 41.0% (by genome).Cells are Gram-stain-negative, strictly aerobic, non-motile, and rod-shaped with 0.5–0.8 μm in width and 1.6–3.9 μm in length. Colonies are yellow, circular, convex, opaque, smooth, and 1–2 mm in diameter after 3 days of incubation at 30 °C on MA. Requires NaT (CGMCC 1.18821T = KCTC 82563T) is isolated from the seawater, collected from the West Pacific Ocean . The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain A3-108T is MW244395 and the GenBank accession number for the whole genome sequence is JAECMS000000000.The type strain A3-108Supplementary file1 (DOCX 3003 KB)Below is the link to the electronic supplementary material.


Fibrotic posterior capsular opacification (PCO), a major complication of cataract surgery, is driven by transforming growth factor–β (TGF-β). Previously, αV integrins were found to be critical for the onset of TGF-β–mediated PCO in vivo; however, the functional heterodimer was unknown. Here, β8 integrin–conditional knockout (β8ITG-cKO) lens epithelial cells (LCs) attenuated their fibrotic responses, while both β5 and β6 integrin–null LCs underwent fibrotic changes similar to WT at 5 days post cataract surgery (PCS). RNA-Seq revealed that β8ITG-cKO LCs attenuated their upregulation of integrins and their ligands, as well as known targets of TGF-β–induced signaling, at 24 hours PCS. Treatment of β8ITG-cKO eyes with active TGF-β1 at the time of surgery rescued the fibrotic response. Treatment of WT mice with an anti-αVβ8 integrin function blocking antibody at the time of surgery ameliorated both canonical TGF-β signaling and LC fibrotic response PCS, and treatment at 5 days PCS, after surgically induced fibrotic responses were established, largely reversed this fibrotic response. These data suggest that αVβ8 integrin is a major regulator of TGF-β activation by LCs PCS and that therapeutics targeting αVβ8 integrin could be effective for fibrotic PCO prevention and treatment.  Cataracts, a major cause of blindness , are treTransforming growth factor–β (TGF-β) signaling mediates the epithelial-mesenchymal transition (EMT) of LCs to myofibroblasts . While TIntegrins, heterodimeric extracellular matrix (ECM) receptors consisting of 1 α and 1 β subunit, mediate cell/ECM attachment, cell migration, and force transmission . IntegriV integrin is critical for the fibrotic response of LCs PCS  and exhibited fibrotic responses, and canonical TGF-β signaling, at 5 days PCS similar to WT, as measured by the expression of a myofibroblast marker, α–smooth muscle actin , and SMAD2/3 phosphorylation . Similarly, systemic administration of either an αVβ5-IBA or αVβ6-IBA to WT mice following fiber cell removal did not alter the fibrotic response of LCs following lens injury  . Overall, this suggests that neither αVβ5 nor αVβ6 integrins are essential for LC EMT PCS.Our prior study revealed that an α LCs PCS . As αV integrins , we soug LCs PCS , and αVβfibrosis , 18–20, 8 integrin subunit was investigated as αVβ8 integrin activates latent TGF-β in some circumstances (8 integrin protein (P ≤ 0.001); this was robust by 3 days PCS (P ≤ 0.001). β8 integrin expression was sustained through 5 days PCS (P ≤ 0.001) though its levels were significantly lower than 3 days PCS. In addition, both αV and β8 integrin levels were similar between WT, β5 integrin–null, and β6 integrin–null capsular bags at 5 days PCS (https://doi.org/10.1172/jci.insight.145715DS1), suggesting that αVβ8 integrin upregulation PCS is not dependent on either αVβ5 integrin or αVβ6 integrin. Further, neither αV nor β8 integrin protein was detected in the intact human lens, but bright αV integrin and β8 integrin staining was detected in LCs exhibiting high levels of α-SMA lining a human capsular bag collected several years after cataract surgery  by mating mice carrying a floxed Itgb8 allele  are transparent  were 3.34% larger in diameter than WT lenses of similar age (P = 0.012), consistent with the phenotype of adult mouse lenses lacking the αV integrin gene (Itgav)  , suggest8ITG-cKO LCs to lens fiber cell removal was tested by following the expression of the myofibroblast marker, α-SMA, and the fibrillar matrix proteins tenascin C and fibronectin  and fibronectin (P = 0.022) protein compared with WT while α-SMA levels remained similar (P = 0.446). WT LCs further upregulated all 3 fibrotic proteins  5 days PCS. In contrast, β8ITG-cKO LCs still expressed less of these proteins 5 days PCS compared with WT , suggesting Itgb8 deletion from the lens inhibits the fibrotic response of remnant LCs PCS.The response of βronectin  PCS , and this downregulation was sustained through 5 days PCS (P ≤ 0.001). In contrast, E cadherin levels were unchanged in β8ITG-cKO 48 hours (P = 0.651) and 5 days PCS (P = 0.390), suggesting that Itgb8-null LCs preserve their epithelial characteristics PCS.We next investigated the expression of an epithelial cell marker, E cadherin, to determine if βours PCS . However8ITG-cKO capsular bags at 5 days PCS are no longer in a monolayer along the capsule, the expression of aquaporin 0, a fiber cell preferred membrane protein, was investigated; some LCs differentiate into structurally aberrant lens fiber cells during PCO pathogenesis, contributing to “pearl-like” PCO when in the visual axis and Soemmering’s ring when restricted to the capsular bag periphery , indicating that β8ITG-cKO LCs can differentiate into lens fiber-like cells PCS to a similar extent as WT.Because the cells associated with βeriphery . Remnantaporin 0  immediat8ITG-cKO capsular bags than WT at 5 days PCS (P ≤ 0.001) (8ITG-cKO mice at any time PCS (data not shown), LC proliferation was investigated by following the expression of Ki67, a marker of all cell cycle stages except G0  and β8ITG-cKO (P = 0.03). However, the WT capsular bag had a higher percentage of Ki67-positive cells compared with β8ITGcKOs at 48 hours PCS (P = 0.02), suggesting β8ITG-cKO LCs proliferate less PCS. These data suggest LCs lacking Itgb8 attenuate their fibrotic response PCS while retaining their epithelial phenotype.Notably, fewer cells were associated with β≤ 0.001) . Becausexcept G0 . At 0 hoxcept G0  in WT or8ITG-cKO LCs manifest by 48 hours PCS, we performed RNA-Seq on WT and β8ITG-cKO LCs at 24 hours PCS to gain insight into this phenotype’s proximal cause. This revealed 2312 genes differentially expressed in WT LCs at 24 hours PCS compared with 0 hours PCS   .Notably, αnt TGF-β  while ge,Itga5) , 28–30, a2, Tnc) , 31, werours PCS . Compariours PCS  revealed-cKO LCs  but 47 gours PCS , suggest8ITG-cKO LCs by assaying the downstream effector of TGF-β signaling, p-SMAD2/3 (P = 0.013) while this induction was attenuated in β8ITG-cKO LCs (P = 0.042). Further, while WT LCs further enhanced this signaling by 5 days PCS (P < 0.001), p-SMAD2/3 levels remained low in β8ITG-cKO LCs (P < 0.001), suggesting that TGF-β signaling PCS depends on the upregulation of β8 integrin expression.Thus, canonical TGF-β pathway activation PCS was determined in WT and β-SMAD2/3   and α-SMA (P = 0.011), tenascin C (P = 0.007), fibronectin (P = 0.012), and collagen I (P = 0.003) 5 days PCS, αVβ8 integrin may play a key role in activating TGF-β signaling in LCs PCS.Because active TGF-β induces LC conversion to myofibroblasts , and β8I defects . As actiVβ8 (Vβ8 integrin function-blocking antibody (ADWA-11), which antagonizes LAP binding to αVβ8 integrin (αVβ8-IBA), thus blocking TGF-β activation  3 days PCS, the time when robust TGF-β signaling is first detected in LCs (P = 0.002), tenascin C (P = 0.003), fibronectin (P ≤ 0.001), and collagen I (P = 0.019) levels. As these attenuations were similar to that observed in β8ITG-cKO LCs (Vβ8-IBA likely blocks TGF-β activation and subsequent fibrotic response of LCs PCS.TGF-β is secreted from cells bound to its latency associated peptide (LAP) and latent binding proteins, forming the latent TGF-β complex . Upon seVβ8 , 14. Thutivation , can inftivation . Systemi-cKO LCs , αVβ8-IBVβ8-IBA works at the level of TGF-β activation by coadministering active TGF-β1 (into the capsular bag) and αVβ8-IBA  to β8ITG-cKO mice at surgery . This confirms that αVβ8-IBA reduces LC fibrosis PCS by blocking the interaction of TGF-β LAP with αVβ8 integrin.We confirmed that α surgery . As expeVβ8-IBA at the time of fiber removal reduces LC fibrosis later PCS  and α-SMA (P ≤ 0.001), tenascin C (P = 0.008), fibronectin (P = 0.025), and collagen I (P = 0.016) levels until at least 5 days PCS. Further, like β8ITG-cKO capsular bags, αVβ8-IBA–treated WT capsular bags exhibited less LC proliferation 3 days PCS  than controls. This supports the idea that blockade of TGF-β activation at the time of lens injury is sufficient to prevent the fibrotic transformation of LCs.Next, we tested if a single treatment with αater PCS . Thus, αVβ8-IBA could arrest or reverse fibrotic PCO once established. Lens fibers were removed from WT mice, and 5 days PCS, they were treated systemically with either a single dose of αVβ8-IBA, or 2 doses of αVβ8-IBA given at 5 and 7.5 days PCS. Samples were harvested 10 days PCS (Vβ8-IBA given 5 days PCS was sufficient to significantly attenuate SMAD2/3 phosphorylation 10 days PCS (Vβ8-IBA-1 dose P = 0.007) to a similar extent as β8ITG-cKO LCs (8ITG-cKO P = 0.009). Similarly, fibrotic marker protein levels were also significantly attenuated in αVβ8-IBA–treated mice 10 days PCS compared with vehicle, and these attenuations were similar to those observed in β8ITG-cKO LCs   .Finally, we investigated whether αdays PCS . Single-Vβ8-IBA given 5 days PCS was insufficient to significantly attenuate α-SMA protein levels (Vβ8-IBA-1 dose P = 0.067). Notably, 2 doses of αVβ8-IBA given 5 and 7.5 days PCS did significantly decrease α-SMA protein levels 10 days PCS (Vβ8-IBA-2 doses P = 0.013). However, α-SMA protein levels were still higher in both treatment groups (Vβ8-IBA -1 dose P = 0.008) and  LCs compared with β8ITG-cKO LCs 10 days PCS. This suggests αVβ8-IBA therapy after LC fibrosis was established halted canonical TGF-β signaling, which substantially reduced fibrosis even in the presence of some degree of EMT.However, single-dose αVβ8 integrin is a core mechanism of PCO development, next we characterized the regulatory relationship between αVβ8 integrin–mediated TGF-β activation and 2 other PCO regulators, gremlin-1 and ECM binding integrins.Because 3 experimental approaches revealed that TGF-β activation by αGrem1 mRNA levels were upregulated 170-fold in WT LCs at 24 hours PCS yet attenuated 3-fold in β8ITG-cKO LCs (P ≤ 0.001) while this upregulation was attenuated in β8ITG-cKO (P = 0.002) and WT (αVβ8-IBA) (P ≤ 0.001) LCs. This trend was also observed 5 days PCS as gremlin-1 protein levels were significantly lower in β8ITG-cKO (P = 0.017) and WT (αVβ8-IBA) (P = 0.022) LCs compared with WT, while the addition of active TGF-β1 to β8ITG-cKO eyes rescued gremlin-1 protein expression compared with β8ITG-cKO (vehicle) (P = 0.004), suggesting that αVβ8 integrin expression by LCs is critical for gremlin-1 upregulation PCS.Previously, we reported that gremlin-1, best known as a BMP antagonist , upregul-cKO LCs . Consist8ITG-cKO mice with recombinant gremlin-1 at cataract surgery did not rescue LC fibrosis  its expression rapidly upregulates in mouse LCs PCS and it is found on fibrotic human LCs at extended times PCS; (b) it can regulate tissue fibrosis and inflammation via binding to the RGD sequence present in the LAP of TGF-β1 and TGF-β3 d TGF-β3 .Vβ8 integrin plays roles in development, fibrosis, inflammation, and wound closure  phenocopies this result. As the addition of active TGF-β1 rescued the attenuated TGF-β signaling and fibrotic responses observed in β8ITG-cKO capsular bags PCS, this suggests that αVβ8 integrin–mediated activation of latent TGF-β is critical for the development of fibrotic PCO. This hypothesis is supported by the observation that treatment of WT mice with ADWA-11, which specifically inhibits the adhesion of LAP to αVβ8 integrin  in concert with the reduction of fibrotic marker mRNAs in β8ITG-cKO LCs at 24 hours PCS, and reductions in fibrotic marker protein expression later PCS, suggest that the αVβ8 integrin/TGF-β signaling axis regulates both EMT and proliferation of LCs PCS, leading to fibrotic PCO.This study explored 3 potential mechanisms by which the V integrins and TGF-β signaling have been previously described  targeting αVβ8 integrin could suffice to prevent TGF-β activation in PCO; 2) the resulting attenuation of fibronectin fibril deposition (likely mediated by α5β1 integrin) may contribute to the long-term prevention of fibrotic PCO, as we have previously reported that fibronectin assembly is required for sustained LC fibrosis PCS  activation has led to intense research into both antibody and small molecule inhibitors of αVβ8 integrin–TGF-β interactions for the treatment (and monitoring) of fibrotic and neoplastic disease  (6 integrin–null mice (tm1DesItgb6) on a 129Svems genetic background were obtained from Xiaozhu Huang (UCSF) (8 integrin subunit gene from the lens (β8ITG-cKO) were created by mating mice harboring an integrin β8 allele on a mixed C57BL/6J 129/Sv background in which exon 4 is flanked by loxP sites   . Homozygg (UCSF) . Mice laom UCSF)  with MLRom UCSF)  (obtaineTransparent lenses (30 ± 2 years of age) were obtained from Lions Eye Bank of Oregon , and aphakic donor eyes were obtained from the Minnesota Lions Eye Bank  as part of their cadaver eye tissue procurement programs. Intact lenses or lens capsular bag/IOL implant complexes were isolated, fresh frozen in OCT medium, and prepared for immunofluorescence experiments as described below.8 gene from the lens was confirmed by PCR analysis of genomic DNA isolated from adult lenses using the primers described in DNA was isolated from tail snips or whole lenses using the PureGene Tissue and Mouse Tail kit (Gentra Systems) as described  and genoLens clarity was determined by viewing isolated lenses using darkfield optics while lens optical properties were assessed by placing lenses on a 200-mesh electron microscopy grid as described previously .Surgical removal of lens fiber cells to mimic human cataract surgery was performed in adult mice as previously described , 67. Bri8ITG-cKO lenses subjected to cataract surgery  were harvested at 0 hours and 24 hours PCS and frozen on dry ice, and RNA was harvested using RNeasy Mini Kit (Samples from WT (C57BL/6) and βMini Kit  from QIAMini Kit . RNA libMini Kit , 25. HeaMini Kit . RNA-Seq8ITG-cKO LCs 24 hours PCS (FDR ≤ 0.05) to discover what proportion of the DEG list had the potential to be influenced by TGF-β signaling the first day PCS.A list of 1603 TGF-β–responsive genes in cultured cells  was filt8ITG-cKO mice immediately following removal of the lens fibers as previously described  or recombinant human gremlin-1 protein  or the BSS vehicle alone into the lens capsular bags of βescribed .Vβ5, αVβ6, and αVβ8 integrin function blocking antibodies was performed as described (Vβ5 integrin blocking) (Vβ6 integrin blocking) (Vβ8 integrin blocking) (Vβ3 integrin that does not cross-react with the mouse αVβ3 integrin protein) at 20 mg/kg in PBS. All the integrin function blocking antibodies were obtained from UCSF.Tail vein injection of αescribed . Brieflylocking) , 3G9 (αVlocking) , or ADWAlocking)  was admiThe details of sample preparation and immunofluorescence were described previously , 72. SupImmunofluorescence images were quantified by determining the MFI of lens capsule–associated tissue viewed in 3 randomly chosen confocal images from biologically independent samples using ImageJ  . The ave8ITG-cKO mice and photographing them in brightfield using a Zeiss STEMI SV 11 dissecting microscope. The diameter of each lens was measured in 2 perpendicular axes using ImageJ, then averaged for statistical analysis.The diameter of adult lenses was determined by dissecting both lenses from 3 WT and 3 βt test (corrected for multiple comparisons using the Holm-Šídák method) or 1-way/2-way ANOVA with Tukey’s post hoc test using GraphPad Prism 8.3.0/9.2.0. Data are presented as mean ± SEM, and differences were considered significant at P ≤ 0.05.All statistics were assessed using either 2-tailed Student’s Animal experiments conformed to the Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Delaware Institutional Animal Care and Use Committee. Experiments using human cadaver-derived lens tissue were reviewed by the University of Delaware Institutional Review Board and were judged as exempt from review.MHS designed research studies, conducted experiments, analyzed data, and wrote the manuscript; SGN conducted experiments and analyzed data; YW designed research studies and conducted experiments; DS provided reagents and designed studies; AA provided reagents and designed studies; TDA provided reagents and designed studies; NMR conducted experiments; APF analyzed data and designed studies; and MKD designed research studies, analyzed data, and wrote the manuscript.


Genetic deletion of the Rictor gene, but not Raptor, in γδ T cells markedly exacerbated liver fibrosis. Mechanistically, CCl4-induced liver injury causes macrophage accumulation in the liver, and IL-1β produced by macrophages promotes mTORC2 signaling activation in γδ T cells, which upregulates T-bet expression and eventually promotes CXCR3 transcription to drive γδ T cell migration. Moreover, hepatic γδ T cells ameliorated liver fibrosis by cytotoxicity against activated hepatic stellate cells in FasL-dependent manner, and secrete IFN-γ to inhibit the differentiation of pro-fibrotic Th17 cells. Thus, IL-1β-activated mTORC2 signaling in γδ T cells upregulates CXCR3 expression, which is critical for IFN-γ+ γδ T cells migration into the liver and amelioration of liver fibrosis. Our findings indicate that targeting the mTORC2 or CXCR3 in γδ T cells could be considered as a promising approach for γδ T cell immunotherapy against liver fibrosis.Liver fibrosis represents a severe stage of liver damage, with hallmarks of inflammation, hepatic stellate cell activation, and extracellular matrix accumulation. Although previous studies demonstrated γδ T cells are involved in liver fibrosis, the precise role and mechanisms of γδ T cells migrating to fibrotic liver have not been elucidated. Here, we aim to investigate the functional subsets of γδ T cells in hepatic fibrosis and to further explore the underlying causes and drivers of migration. In this study, we observed that γδ T cells accumulate in fibrotic liver. Adoptive transfer of γδ T, especially Vγ4 γδ T subset, can significantly alleviate liver fibrosis. In addition, CCl Fibrosis occurs in almost every part of the body and causes organ failure in the context of most chronic diseases . The damImmune cells and immune responses are critically involved in liver fibrosis , 4. Duriγδ T cells, although expressing TCRs, are characterized as innate immune cells and exert critical functions in diseases . γδ T ceMammalian/mechanistic target of rapamycin (mTOR) complexes are important orchestrators of the metabolism of immune cells, governing cell differentiation and function . Previou+ γδ T cells accumulation in fibrotic liver remains unknown.Although it is known that γδ T cells produce IFN-γ to inhibit the liver fibrosis , it is n+ γδ T cells to migrate to the fibrotic liver through upregulating of CXCR3. Thus, we found previously unappreciated mechanisms of γδ T cells migrate into the liver and exert a protective effect against liver fibrosis. Targeting mTORC2 pathways may provide important clues for developing therapeutics to inhibit liver fibrosis.In this study, we found that γδ T cell produced IFN-γ inhibited pro-fibrotic Th17 cells in liver fibrosis. And macrophage-derived IL-1β contributes to mTORC2 activation of γδ T cells, which is required for IFN-γtm1.1Dmsa/J (B6 Raptor-flox), Rictortm1.1Klg/SjmJ (B6 Rictor-flox), B6.Cg-Tg (CD2-icre) 4Kio/J (hCD2-iCre), B6.129S7-Ifngtm1Ts/J (IFN-γ-/-), C57BL/6-Il17atm1Bcgen/J (IL-17A-/-), B6.129S4-Ifngtm3.1Lky/J (IFN-γ-eYFP), C57BL/6-Il17atm1Bcgen/J (IL-17-GFP), B6.129P2-Cxcr3tm1Dgen/J (CXCR3-/-), B6.129S6-Tbx21tm1Glm/J (Tbx21-/-) and B6.129P2-Tcrdtm1Mom/J (TCR δ−/−) mice were purchased from The Jackson Laboratory . CD45.1 mice were given by Zhongjun Dong from Tsinghua University . Sex- and age-matched animals were randomly assigned to different groups. All mice were maintained under SPF conditions and all animal procedures were approved by the Institutional Animal Care and Use Committee of Jinan University.B6.Cg-RptorRecombinant mouse (rm)IL-2 (Peprotech), purified anti-mouse CD3 mAb (145-2C11), anti-mouse CD28 mAb (PV1), hamster anti-mouse TCR Vγ1 mAb (2.11), and hamster anti-mouse TCR Vγ4 mAb (UC3) were from Sungene Biotech . FITC-conjugated anti-mouse CD4 (RM4-5), and PE-conjugated anti-mouse γδ TCR (GL3), APC-conjugated anti-mouse NKG2D (CX5), eFlour450-conjugated anti-mouse CD3 (17A2), Percpcy5.5-conjugated anti-mouse IL-17 mAb (eBio17B7) and Foxp3 staining buffer set were purchased from eBioscience . APC/Percpcy5.5-conjugated anti-mouse CD4 (GK1.5), FITC/APC-conjugated anti-mouse TCR γδ mAb (GL3), PE-Cy7-conjugated anti-mouse IFN-γ mAb (XMG1.2), PE-Cy7-conjugated anti-mouse CD3 mAb (145-2C11), APC-conjugated anti-mouse Vγ4 mAb (UC3-10A6), PE anti-mouse CXCR3 mAb (CXCR3-173), Percpcy5.5 anti-mouse CCR3 mAb (J073E5), Alexa Fluor 647 anti-mouse CCR5 mAb (HM-CCR5), Brilliant Violet 510 anti-mouse CCR2 mAb (SA203G11), PE-Cy7 anti-mouse CXCR5 (L138D7) and PE-Cy7 anti-mouse CXCR6 (SA051D1), Alexa Fluor 647 anti-mouse F4/80 mAb (BM8), and PE anti-mouse Ly6G mAb (1A8) were purchased from Biolegend . PE-conjugated anti-mouse p-Akt473 mAb (D9E), Alexa Fluor 647- conjugated anti-mouse p-Akt473 mAb (D9E) and Alexa Fluor 488-conjugated anti-human/mouse p-S6 mAb (D57.2.2E) were purchased from Cell Signaling Technology . PMA/ionomycin were purchased from Sigma, Inc. , GolgiStop was purchased from BD Biosciences , Anti-alpha smooth muscle Actin antibody was purchased from Abcam .4   or vehicle (corn oil) three times a week for 4 weeks. For toxic acute liver injury, mice were given i.p. injection of a double dose of CCl4, and the mice were euthanized 48 h after CCl4 injection. For genetic mouse experiments, age- and sex-matched WT and TCRδ−/− mice were co-housed at a 1:1 ratio for at least 4 weeks to minimize the potential microbiome effects. The sample size was chosen to ensure the possibility of statistical analysis and to also minimize the use of animals in accordance with the animal experiment committee of Jinan University. The results from previous results were also used to determine the sample size.For induction of liver fibrosis, 6–8-week-old male mice were injected intraperitoneally (i.p.) with CCl2, 4 mM MgSO4, and 10 mM 4-[2-hydroxyethyl]-1-piperazine ethane sulfonic acid). The liver was cut into 2 mm3 pieces and shaken at 100 rpm for 30 min in a 37 °C incubator. Single-cell suspensions were filtered through a 70-µm cell strainer, then fractionated with 30% Percoll (Sigma) at 1.04 g/mL, NPCs were resuspended in erythrocyte lysis buffer, and washed with HBSS. The NPCs suspension was used for flow cytometric analysis.Isolation of liver nonparenchymal cells (NPCs) was performed following an established method . In briet-test between two groups or one-way ANOVA for comparison of two or multiple groups, respectively. The following terminology is used to show statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001. No samples or animals were excluded from the analysis. Statistical analysis was performed with GraphPad Prism software. Other detailed materials and methods are described in the All quantitative data were shown as mean ± SD unless otherwise indicated. All samples were compared using two-tailed, unpaired Student’s -/-), by repetitive CCl4 treatment. WT mice showed more inflammatory responses in the liver  displayed significantly enhanced liver fibrosis, as assessed by H&E, Sirius Red and Masson’s Trichrome staining  and p-AKT(pS473), respectively. Interestingly, p-S6(ser235/236) was slightly activated in hepatic Vγ1 cells, and p-AKT(pS473) was conspicuously activated in hepatic Vγ1 and Vγ4 cells  and Rictor-f/f-CD2-Cre (RicKO) mice were sorted for RNAseq analysis. γδ T cells from RicKO showed that the expression of several chemokine receptors reduced, including CCR9, CXCR3, CCR2, CCR10, CXCR6, CCR4 and CCR6  and Rictor-f/f-CD2-cre (RicKO) Vγ4 cells into TCRδ-/- mice respectively and then induced liver fibrosis. The results showed that Vγ4 cells with Raptor deficiency reconstituted mice had no significant effect on the progression of liver fibrosis compared with Rapf/f Vγ4 cells reconstituted mice  to exert cytotoxicity against activated HSCs, and ultimately protects the liver from fibrosis (Fig. Collectively, we found that mTORC2 signaling, which can be activated by macrophage-derived IL-1β in serum, potentiates CXCR3 expression in Vγ4 cells after CClLiver fibrosis/cirrhosis is characterized by a complex inflammatory response with tightly regulated interactions between damaged hepatocytes and recruited immune cells , 22. IFN-/- mice was associated with reduced IFN-γ expression and a reduction of IFN-γ–positive intrahepatic T cells [+ γδ T cells recruited to the injured liver; subsequently, our results demonstrated that CXCR3 and its ligands regulate the migration of IFN-γ+ γδ T cells. These data suggested that CXCR3 contributed to an increased number of IFN-γ+ γδ T cells that were observed during liver fibrosis.Interestingly, naturally occurring IFNγ-producing Th1 cells are almost exclusively CXCR3 positive . In addi T cells . Hence, mTOR endows T cells with the ability to properly sense and integrate diverse environmental signals including nutrients and growth factors , and was+ γδ T cells to the fibrotic liver in a CXCR3 dependent manner (Fig. Taken together, our study revealed that mTORC2 activation of γδ T cells is dependent on IL-1β producing hepatic infiltrated macrophages, which are important for the migration of IFN-γner Fig. . MoreoveSupplementary InformationChecklist


Solid-state photoreaction did not occur in the solid-state but it occurred in acetonitrile solution.The photoreactive behaviour of the title compound, C N,N-bis(propan-2-yl)acetamide], C20H23NO2 was synthesized and its photoreactive properties in the crystalline state and in acetonitrile solution were investigated. The compound crystallizes in the chiral space group P212121. The crystal does not react under UV light irradiation, perhaps due to the presence of the biphenyl group. However, the compound is photoreactive in acetonitrile solution to give racemic 3-(p-phenylphenyl)-3-hydroxy-N-isopropyl-4,4-dimethylazetidin-2-one.The title compound [systematic name: 2--2-oxo- N,N-dialkyl-α-oxoamides has been studied in relation to penicillin chemistry glyoxylamides 1b and 1c also form chiral crystals, and photoirradiation in the solid state gives optically active β-lactam derivatives 2b and 2c, respectively glyoxylamide 1b and 1c do not form chiral crystals, and their photoirradiation in the solid state gives racemic β-lactam derivatives 2b and 2c, respectively. Therefore, we synthesized the novel title compound 1d having a phenyl group and investigated whether optically active β-lactam derivative 2d could be obtained by photoreaction. It was found that 1d formed a chiral crystal in the chiral space group P212121, but photoreaction did not proceed in the solid state. However, photoreaction of 1d in acetonitrile solution proceeded to give racemic 3-(p-phenylphenyl)-3-hydroxy-N-isopropyl-4,4-dimethylazetidin-2-one 2d in 26% yield. In this study, although 1d formed a chiral crystal, the reason why the photoreaction product of 1d in the solid state was not obtained was clarified by single-crystal X-ray structural analysis, UV spectroscopy and time-dependent density functional theory (TDDFT) calculations.The solid-state photochemistry of 1a are 88.0 (4) and −5.1 (4)°. In order for the Norrish–Yang reaction to take place, the reacting atoms in the molecular structure must be in close proximity. The Yang cyclization of α-oxoamides to β-lactams starts with abstraction of the γ-hydrogen (with respect to the benzylic carbonyl) by the benzylic carbonyl oxygen in the excited state. In the title compound, there are two γ-hydrogen atoms (H5 on C9 and H12 on C12). The distances between the carbonyl oxygen atom O1 and the respective γ-hydrogen atoms H5 and H12 are 2.65 and 5.01 Å. The former interatomic distance is within the ideal value of up to about 2.7 Å, at which photoreaction can proceed in the crystal  level ] method. In the calculated UV–vis spectra, there were two weak peaks at 254 and 362 nm for 1a, and there was an intense and broad peak at 310 nm for 1d. The calculated spectra were similar to the experimental spectra , and C—H⋯O  interactions forming a 1D zigzag chain structure along the b-axis direction . Details of these interactions are given in Table 1In the crystal, the molecules are linked by weak intermolecular C—H⋯O  interactions forming a 1D chain structure along the on Fig. 6a, and Con Fig. 6b. Detaiet al., 2016N,N-diisopropylphenylglyoxylamide fragment shown in Fig. 1et al., 1989et al., 2005et al., 1995et al., 1996A search of the Cambridge Structural Database acetic acid with thionyl chloride followed by reaction with N,N-diisopropylamine. Thus, to an ice-cooled solution of N,N-diisopropylamine  in dry diethyl ether (45 mL) was added a solution of 4-phenylbenzoylformyl chloride  in dry diethyl ether (45 mL), and the reaction mixture was stirred for 10 h at room temperature. After filtration of N,N-diisopropylammonium chloride, the filtrate was washed with dilute HCl and aqueous NaHCO3 and dried over MgSO4. The crude product was purified by silica gel column chromatography (toluene:ethyl acetate = 9:1) and recrystallized from toluene to give 1d as colorless prisms ; IR (KBr): νmax 1640, 1680 cm−1; 1H NMR : δ 8.01 , 7.73 , 7.63 , 7.50–7.39 , 3.75 , 3.61 , 1.60 , 1.20 ; 13C NMR : δ 190.7, 167.0, 147.1, 139.7, 132.1, 130.1, 129.0, 128.5, 127.6, 127.4, 50.2, 46.1, 20.6, 20.4; ESIMS m/z: calculated for C20H23NNaO2 [M + Na]+, 322.1621; found, 322.1586. Single crystals of 1d suitable for X-ray diffraction analysis were grown from a toluene solution.The title compound was prepared according to a reported method  was pulverized in a mortar and irradiated with a 400 W high-pressure mercury lamp for 300 h. No reaction took place, as determined by TLC, IR and NMR spectroscopies. 1d  in acetonitrile (10 mL) was irradiated with a 400 W high-pressure mercury lamp for 60 h. The crude product was purified by silica gel column chromatography (toluene:ethyl acetate = 4:1) to give 3-(p-phenylphenyl)-3-hydroxy-N-isopropyl-4,4-dimethylazetidin-2-one 2d as a colorless powder ; IR (KBr): νmax 3200, 1720 cm−1; 1H NMR : δ 7.60–7.00 , 4.64 , 3.57 , 1.44 , 1.27 , 0.87 ; ESIMS m/z: calculated for C20H23NNaO2 [M + Na]+, 322.1621; found, 322.1569.Uiso(H) = 1.2Ueq(C) and 1.5Ueq(C-methyl). The Flack parameter x is 0.1 (4) as shown in Table 2et al., 2008et al., 2013PLATON  I. DOI: 10.1107/S2056989021005387/dj2024Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021005387/dj2024Isup3.cmlSupporting information file. DOI: 2085220CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-021-96703-y, published online 03 September 2021Correction to: The original version of this Article contained an error in Table 2, where the “Ascorbic acid group, n of outcomes/total no-of patients” for “Ascorbic Acid” was incorrect for the Outcome “Thrombosis during ICU, Analysis on all eligible patients”.“9/5.8 (2.3)”.now reads:“9/154 (5.8)”.The original Article has been corrected.


PThe title compound was synthesized from a dinitrobiphenylbenzene derivative using a novel modification of the Cadogan reaction. The reaction has several possible ring-closed products and the title compound was separated as the major product. It crystallizes in the monoclinic  18H12N2O2, was synthesized from a dinitrobiphenylbenzene derivative using a novel modification of the Cadogan reaction. The reaction has several possible ring-closed products and the title compound was separated as the major product. The X-ray crystallographic study revealed that the carbazole compound crystallizes in the monoclinic PThe title compound, C This N-containing heterocyclic compound was discovered by Graebe and Glaser in 1872  = 3.3963 (9) Å and Cg8⋯Cg8 = 3.3982 (10) Å where Cg1 and Cg8 are the centroids of the N1/C1/C6/C7/C12 and N3/C19/C24/C25/C30 rings, respectively] with adjacent carbazole rings within the stacks being almost parallel. The combination of hydrogen bonding and π-stacked carbazole ring systems results in the formation of a three-dimensional interaction.In the crystal, the two molecules are linked into [110] chains by N—H⋯O and N—H⋯N hydrogen bonds involving the carbazole N atom of one independent molecule and the nitro group of the other Table 1, as showet al. 2016et al., 2004p-tolyl-9H-carbazole-3-carbonitrile; Ramathilagam et al., 2011H-carbazole; Li et al., 2006H-carbazole-1,3-dicarboxalate; Verma et al., 2015H-carbazole; Matsubara et al., 2016A search of the Cambridge Structural Database  to 1.4773 a.u. (blue) as shown in Fig. 3i.e. negative dnorm values on the surface, which highlight the hydrogen-bonding interactions.A Hirshfeld surface analysis   and 0.655 g (2.5 mmol) of triphenylphosphine. 8 mL of the solvent o-dichlorobenzene were added o the reaction mixture. The resulting reaction mixture was stirred at 473 K under nitrogen for 24 h. The solvent was removed under reduced pressure at 333 K and the crude product was purified by column chromatography  to provide 0.230 g of the title product as a beige solid (yield: 86%). 1H NMR : δ 8.39 , 8.09 , 8.05 , 7.56–7.51 , 7.48–7.38 , 7.32 .The synthesis of the title compound is shown in Fig. 5Uiso(H) = 1.2Ueq(N). C-bound H atoms were positioned geometrically (C—H = 0.95 Å) and refined as riding with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021007726/ex2047sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021007726/ex2047Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021007726/ex2047Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021007726/ex2047Isup4.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021007726/ex2047Isup5.cmlSupporting information file. DOI: 2099768CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Analytic formulas were developed to explore the new features, including: (i) both free radical photopolymerization (FRP) efficacy and the production of nanogold (NG), which are proportional to the relative concentration ratios of (A0 + B0 + N0)/G0 and may be optimized for maximum efficacy; (ii) the two competing procedures of NG production and the efficacy of FRP, which can be tailored for an optimal system with nanogold in the polymer matrix; (iii) the FRP efficacy, which is contributed by three components given by the excited state of copper complex (T), and the radicals (R and S) produced by iodonium and amine, respectively; (iv) NG production, which is contributed by the coupling of T and radical (S) with gold ion; and (v) NG production, which has a transient state proportional to the light intensity and the concentration ratio A0/G0) + (N0/(K’M0), but also a steady-state independent of the light intensity. This article presents, for the first time, the kinetics and the general features of a photopolymerization system (under visible light), copper-complex/Iodonium/triethylamine/gold-chloride (orA/B/N/G), with initial concentrations of A Light sources from UV 365 nm) to near-infrared 980 nm) have been used for photopolymerization reactions in many industrial and medical applications such as dental curing, microlithography, stereolithography, microelectronics, and holography ,2. A var65 nm to 0 nm haveN-vinylcarbazole (NVK) was investigated by Mokbel et al. *, B = Iod; N is triethylamine (TEA), R = [HLCuCl]The kinetic equations for the concentration of each component of the 3-components (A/B/N) system shown in  are consE is the regeneration (REG) term of of the initiator, [A], given by RE = g(k7 + k1[B]), with g = k7 + k1[B] + k2[N] + k3G. b = 83.6a’wq, where w is the light wavelength (in cm) and q is the triplet state T quantum yield; a’ is the mole absorption coefficient, in (1/mM/%) and I  is the light intensity, in mW/cm2. All the rate constants are defined previously [j  are for the couplings of T and [B], [N], and G, respectively; K is for the coupling radicals R with monomer M (for FRP). In the above kinetics, we include the bimolecular termination  [B]), S = k2bIgg”[A] [N]), G’ = (k3T + k4S)/(k5T+k6S); with g = 1/(k7 + k1[B] + k2[N] + k3G + k5G’ + kM), g’ = 1/(k”S + KM), g” = 1/(k4G + k6G’ + k”R + K’M). For comprehensive modeling, we will use the so-called quasi-steady state assumption ,26,27. TUnder the above quasi-steady-state solutions, we obtain the simplified equations as follows.The monomer conversions for FRP and generation of NG are given by (15) dMdA full numerical simulation is required for the solutions of Equations (11)–(16), which will be presented elsewhere. We will focus on comprehensive analysis for special features and the key factors for efficient producing of NG related to the measured data of Tar et al. , based o4G such that g = 1/(k3G), g’ = 1/(KM), and g” = 1/(K’M). In addition, RE is taken as a mean reduction factor (f’), such that (1–RE) = f’ = [1 − k2N0/(k3G0)] is time independent, having a value of f’ = 0.5 to 1.0. The first-order solutions of Equation (11) to (15) are found: [A] = A0 exp(–dt); with d = f’bI; [B] = B0 exp(-H), with H(t) = DE(t), with E(t) = [1 – exp(–dt)]/d]; [N] = N0 exp(–H’), H’ = D’(k2/k1)D, with D = (k1/k3)d(A0/G0). Also G(t) = G0 – (k1/k3)dt; G’ = (k3/k5)[1 + k4S/T] = (k3/k5)[1 + k4[N]/(KM), for k5T >> k6S. Using these approximated solutions, Equations (15) and (16) become3G). Analytic formulas need assumption of strong coupling of T and [B], and KM >> k”S, K’M >> k0, and G’ = (k3/k5).3)bI(A0/G0); and Ho(t) is a complex second order term proportional to bIA0(k1B0 + k2N0/B0)/G0. We note that P(t) has a transient state value P = Qt, and steady-state value, P = Q/d, which is independent of the light intensity. Solving Equation (17), we obtain the first-order solution, with G(t) = G6k2/k3)(N0/(K’M0), E(t) = [1– exp(–dt)]/d]; E’(t) = [1– exp(–d’t)]/d’]; D’ = bI(A0/G0); d’ = d + (k1/k3)D, D = (k1/k3)d(A0/G0). We note that [NG](t) has a transient state value [NG] = D’(1 + Q’t); and steady-state value, [NG] = D’(1/d + Q’/d’), which is independent to the light intensity. The production of nanogold, [NG], is given by the solution of Equation (18),As shown by Equations (17)–(20), the following significant features of the A/B/N/Gold, or Cu/Iod/Amine/Gold, system are summarized as follows  term in Equation (11). (i) The additive [B], or Iod (an oxidizing agent) interacts with copper excited state  to produce radical (R) and also to regenerate the initiator, [A] (or Cu), shown by R0) which leads to FRP; and coupled with G, or Au(3+), to form Au(2+), then G’, or Au(1+), leading to Au(0) and the nanogold (NG). (ii) The additive, [N] (or amine), has dual functions: interacting with T to produce additional radical /(k3G0). which is an increasing function of the absorption coefficient (b) and light intensity (I), and the concentration ratios of (A0 + B0+ N0)G0. (iii) Equations (9) and (17) show that the FRP is contributed by three components: from the coupling of monomer (M) with T, R, and S. The conversion efficacy is proportional to bI(A5T + k6S)G’ shown by Equation (10). In addition, as shown by Equation (20), [NG](t) has a transient state value [NG] = D’(1 + Q’)t, which is an increasing function of bI = D’(1/d + Q’/d’), which is independent to the light intensity. (iv) Equations (10) and (18) show that the production of NG is contributed by two components given by the coupling term of /G0, rather than the individual concentrations. Therefore, the photoinitiating system reported by Tar et al.  plays no role in the production of NG under the first-order solution, but plays a minor role reducing the NG production, as shown by our second-order factor g = (1 − k0 + B0 + N0)/G0, which has an optimal value with nanogold in the polymer matrix.Analytic formulas are developed to explore the kinetics of a G1/ Iodonium/ TEA/gold chlorid (or A/B/N/G) system. We found that the FRP efficacy is governed by the coupling of the excited state of copper complex (T) with the radicals (R and S) produced by iodomium and amine, respectively. NG production has a transient state proportional to the light intensity, whereas it is independent to the light intensity at steady-state. The competing between NG production and efficacy of FRP can be tailored by the relative concentration ratios of (A


The terminal phenyl rings are almost parallel to each other [dihedral angle 21.71 (10)°]. Both of these rings are orthogonal to the central phenyl ring, subtending a dihedral angle of about 78° in each case. The extended structure is consolidated by C—H⋯O and C—H⋯S hydrogen bonds as well as aromatic ring interactions of parallel-displaced and T-type. The molecule has approximate C2 local symmetry but this is not carried over to its three-dimensional structure or the intermolecular interactions.In the racemic title compound, C Each of these ‘bookend’ rings is orthogonal to the central phenyl ring subtending dihedral angles of 78.50 (9) (C5/C11 rings) and 78.80 (9)° (C11/C21 rings). The thiazine ring containing atom S1 exhibits a twisted-boat pucker  while the ring containing S2 has a half-chair pucker  with atom S2 displaced from the plane defined by the remaining five atoms of the ring, by about 0.968 Å. Neither of these puckers are of the most favored type. Despite the different conformations of the heterocyclic rings, the molecule possesses approximate local C2 symmetry with an r.m.s. deviation for the overlapping halves of the molecule of 0.261 Å but this local symmetry does not coincide with any crystallographic symmetry in the lattice. Consequently there is asymmetry in the intermolecular interactions  alternating with two 1,3-thiazine rings, C1–C4/N1/S1 and C17–C20/N2/S2 Fig. 1. In the ns Fig. 2.a-axis direction  act as acceptors for C—H⋯O and C—H⋯S type intermolecular interactions Table 1. The donon Fig. 3 shows la al. 2020 have recet al., 2016Chemical context section above, only our prior paper reports a crystal structure bis-2-(3-nitrophenyl)-3-phenyl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one and rac-2-(4-nitrophenyl)-3-phenyl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one -4-oxo-2,3-diphenyl-1,3-thiazinan-5-yl]acetamide -7,8,9-trihydroxyhexahydro-4H,6H-pyridothiazin-4-one -6-methoxy-4-oxo-2-(prop-1-en-2-yl)-1,3-thiazinane-2-carboxylate  was performed in the Cambridge Structural Database dianiline  and 3-mercaptopropionic acid  were added. 2-Methyltetrahydrofuran (2.3 ml) was added and the solution was stirred. Pyridine  was added. Finally, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P) in 2-methyltetrahydrofuran  was added. The reaction was stirred at room temperature and followed by TLC, then poured into a separatory funnel with dichloromethane (20 ml). The mixture was washed with water (10 ml). The aqueous fraction was then extracted twice with dichloromethane (10 ml each). The organics were combined and washed with saturated sodium bicarbonate (10 ml) and then saturated sodium chloride (10 ml). The organic fraction was dried over sodium sulfate and concentrated under vacuum to give a pale yellow crude solid. Recrystallization from ethanol solution gave two crops of off-white solid . m.p. 476.3–483.7 K (decomp.). Crystals suitable for X-ray analysis were grown by slow evaporation from ethanol solution.A two-necked 25 ml round-bottom flask was oven-dried, cooled under NCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021011592/hb7986sup1.cifCrystal structure: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021011592/hb7986Isup3.cmlSupporting information file. DOI: 10.1107/S2056989021011592/hb7986Isup4.hklStructure factors: contains datablock(s) I. DOI: 2119921CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


E)-imino]methyl}phenol was synthesized by an eco-friendly microwave-assisted method that is highly selective and efficient. In the crystal, molecules are linked by N—H⋯N and C—H⋯O hydrogen bonds.2-{[-imino]methyl}phenol in good yields and purity. The title compound is a Schiff base that exists in the phenol–imine tautomeric form and adopts an E configuration. The three independent molecules in the asymmetric unit  are not planar, the cyclobutyl and the phenol-imine rings are twisted to each other making a dihedral angle of 67.8 (4)° in molecule A, 69.1 (2)° in molecule B and 89.1 (2)° in molecule C. In each molecule an intramolecular O—H⋯N hydrogen bond is present, forming an S(6) ring motif. A Hirshfeld surface analysis was performed to investigate the contributions of the different intermolecular contacts within the supramolecular structure. The major interactions are H⋯H (53%), C⋯H (19%) and N⋯H (17%) for molecule A, H⋯H (50%), N⋯H (20%) and C⋯H (20%) for molecule B and H⋯H (57%), C⋯H (14%) and N⋯H (13%) for molecule C.The title compound, C In this work, an eco-friendly protocol for the synthesis of Schiff bases from 3-amino-5-cyclobutyl-1,2,4-triazole and salicylaldehyde in ethanol under microwave irradiation was developed. In addition, 2-[(E)-methyl]phenol was characterized by single crystal X-ray diffraction and investigated using Hirshfeld surface analysis.Imines (Schiff bases) have been extensively used as analytical and medicinal materials (Bülbül A. The cyclobutyl ring is twisted by 73.9 (3) and 67.8 (4)°, with respect to the 1,2,4-triazole, and phenol-imine rings in molecule A. The corresponding angles in molecule B are 18.7 (3), 74.6 (2) and 69.1 (2)° and 3.2 (4), 85.9 (2) and 89.1 (2)° for molecule C. When these angles for the three molecules are compared, it is observed that there is a harmony between them, as well as significant differences, especially in the angles between the phenol-imine and 1,2,4-triazole rings [3.2 (4)° in molecule C but around 18° in A and B] and the phenol-imine cyclobutyl rings . In the molecules, the C=N group has a strong electron-withdrawing character, as revealed by the double-bond character of the C=N bond [1.276 (6)–1.287 (6) Å] and the single-bond character of C—O [1.349 (5)–1.355 (6) Å] in the phenol–imine tautomer. Furthermore, the azomethine C=N double bond has an E configuration. These values and other bond lengths and angles  ring motif.The molecular structure of the title compound, (I)s Table 1 are in gD⋯N10ii, N7—H7D⋯N2, N11—H11⋯N6 and C10—H10⋯O3i -N-benzylidene-1H-1,2,4-triazol-5-amine as the main skeleton, viz. 5-methyl-2-[methyl]phenol methanimine imino]methyl}phenol methyl]phenol methyl]-2-naphthol  ring motif as in the title compound. The two benzene rings (1 and 3) and the triazole ring (2) in WEFTUX, are almost in the same plane, the angles between rings 1 and 2, and between rings 2 and 3 being 3.7 (2) and 3.3 (2)°, respectively. This latter angle is 4.58 (8)° in PEVXAS. In the structures mentioned above, the twist angles between triazole and phenyl rings are quite small, as in molecule C of (I)A and B of the title compound, these angles are over 18°. All compounds were isolated as the phenol-imine (O—H⋯N) tautomeric form, as in (I)H-1,2,4-triazole derivatives mentioned above.There are no direct precedents for the structure of (I)CrystalExplorer17  to 1.5176 Å (blue) (molecule A), −0.6084 (red) to 1.2881 Å (blue) (molecule B) and −0.6060 (red) to 1.5351 Å (blue) (molecule C), respectively. In Fig. 3normd surface of molecules A, B and C represents the N—H⋯N interactions. The major interactions of the compound , N⋯H (20%) and C⋯H (20%) for molecule B and H⋯H (57%), C⋯H (14%) and N⋯H (13%) for molecule C. It was found that the structure is stabilized by hydrogen bonds .We performed a Hirshfeld surface analysis and generated the associated two-dimensional fingerprint plots  was added. The mixtures were irradiated at 393 K for 30 min. The precipitated solid was filtered, washed with cold ethanol and dried at 353 K. The title compound was obtained in the form of a pale-yellow solid in 92% yield. It was recrystallized from ethanol (m.p. 448–449 K). The reaction scheme is shown in Fig. 4Salicylaldehyde (1.0 mmol), 3-amino-5-cyclobutyl-1,2,4-triazole (1.0 mmol) and absolute EtOH (2.0 ml) were mixed in a microwave process vial (10 ml), then a 4 −1): 759, 991, 1030, 1076, 1276, 1562, 1612, 2986, 3040.IR : 13.73 , 12.60 , 9.34 , 7.90–6.80 , 3.70–3.30 , 2.55–1.75 .13H14N4O. Calculated, %: C, 64.45; H, 5.82; N, 23.13.Elemental analysis : Found, %: C 64.31; H 5.64; N 23.79. CUiso(H) = 1.5Ueq(O). The N-bound H atom was located in a difference-Fourier map. Its parameters were adjusted to give N—H = 0.86 Å and it was then refined as riding with Uiso(H) =1.2Ueq(N). The C-bound H atoms were positioned geometrically and refined using a riding model with C—H = 0.93 Å and Uiso(H) = 1.2Ueq(C) for aromatic and other H atoms, and with C—H = 0.97 Å and Uiso(H) = 1.5Ueq(C) for methylene H atoms. The crystal studied was refined as a two-component inversion twin.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021011658/zv2010sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021011658/zv2010Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021011658/zv2010Isup3.cmlSupporting information file. DOI: 2120120CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Here we show that a hybrid BN/carbene approach allows access to an unsymmetrical analogue of Thiele’s hydrocarbon 1, and that this strategy confers markedly enhanced stability on the radical cation. 1+• is stable across an exceptionally wide redox range (> 1 V), permitting its isolation in crystalline form. Further single-electron oxidation affords borenium dication 12+, thereby establishing an organoboron redox system fully characterized in all three redox states. We perceive that this strategy can be extended to other transient organic radicals to widen their redox stability window and facilitate their isolation.Thiele’s hydrocarbon occupies a central role as an open-shell platform for new organic materials, however little is known about its redox behaviour. While recent synthetic approaches involving symmetrical carbene substitution of the CPh Organic molecules that can access various redox states have potential applications in electronics, batteries, catalysis, among others. Here the authors report the preparation of an unsymmetrical organoboron analogue of Thiele’s hydrocarbon and study its one- and two-electron oxidation reactions; remarkably, the radical cation is stable over a redox range of > 1 V and can also be isolated. Subsequently, its X-ray structure was determined, confirming its diradicaloid character3. Since then, Thiele’s hydrocarbon has been widely exploited and integrated into the design of new organic materials with small HOMO – LUMO energy gaps4.Shortly after Gomberg’s pioneering discovery of the persistent triphenylmethyl radical in 190014, reflecting applications as super-electron donors15, photo-/electro-catalysts16, redox switches17, or in redox-flow batteries etc18. However, such systems are rarely isolated, due to the redox instability of the intervening radical species13. Thiele’s hydrocarbon, which can gain aromaticity upon successive one-electron oxidations, presents itself as a privileged platform for such applications. However, its redox properties are far less studied, restricted by the transient nature of its radical monocation (t1/2 = 3 s), and structural information for both its mono- and dication are unknown  approaches which necessitate the use of multiple planar-fused π-conjugated rings to facilitate spin delocalization4. Importantly, the diverse library of available carbenes also allows for modular tuning of the CPh2 termini32. That said, recently reported analogues based on N-heterocyclic carbenes (NHCs) by Ghadwal et al., cyclic (amino)carbenes (CAACs) and acyclic diaminocarbenes (ADCs) by Jana et al. are characterized either by two single-electron transfer events at potentials which are very close (ΔE < 0.25 V)29, or by a single two-electron redox wave31, alluding to the instability of the respective mixed-valence radical cations towards disproportionation33. As such, these systems are typically limited to only two stable redox states, viz. the neutral and dicationic forms. Most recently, Jana et al. pushed this strategy further, reporting that symmetrical CH(NDippMe) disubstitution accesses a more stable radical cation with a redox range extended to 0.48 V34.Carbenes have recently emerged as highly efficient ligands to stabilize organic radicals36, leading to the synthesis of silicon37, mono-/dianionic boron39, and dicationic nitrogen analogues of Thiele’s hydrocarbon40. In regards to anionic boron analogues, three-state redox systems have been reported very recently, but the redox ranges associated with the intermediate radicals are also very narrow (ΔE = 0.20 V), thus limiting the stability of these radicals.Main group heteroatoms have been introduced to modify the electronic properties of π-conjugated organic molecules41. Interestingly, this approach has recently been shown to enhance the electronic coupling in electron-rich olefins, leading to isolable olefin radical cations, albeit at the expense of decreasing the stability of the dicationic species, meaning that a three-state redox system could not be achieved42. In the context of systems based on Thiele’s hydrocarbon, replacement of both exocyclic C=C double bonds with B=N successfully delivers isolable BN analogues, as reported by the groups of Piers, Yamashita and Ye  would be expected to render the intervening radical cation stable across a wide redox range by disfavouring the second oxidation step (cf. bis-BN systems), albeit moderated somewhat by the presence of the opposing NHC function (Method A) which would mitigate the enhanced electrophilicity at boron in the doubly oxidized dication55.Inspired by these reports, we hypothesized that an unsymmetrical mono-BN analogue of Thiele’s hydrocarbon featuring isoelectronic NHC and N-heterocyclic boryl (NHB)2BOTf and IDipp leads to the formation of a B/C frustrated Lewis pair (FLP) system , and in this case crystallographic study unambiguously confirms the identity of 2 (and provides credence to 1) as the target unsymmetrical organoboron analogue of Thiele’s hydrocarbon.The large steric profile inherent in both triflate-substituted borane (HCDippN)tem Fig. 56. Upon 2 adopts a curved shape . Moreover, consideration of the natural bond orders obtained from NRT  calculations, suggests that the predominant resonance structure features exocyclic B–N single and C=C double bonds  falls within the range of bis-carbene systems  occurs at a much more positive potential, leading to a markedly wider separation (ΔE = 1.08 V) between the two redox events.Cyclic voltammetry was used to elucidate the redox properties of ure Fig. . The fir1+• not only greatly exceeds that of the elusive radical cations of bis-carbene analogues 39 reported by Jana et al. and the pyridyl-carbene radical cation (ΔE = 0.59 V)12 reported by Hansmann et al. — both of which exhibit much narrower redox ranges independently.The redox range associated with radical cation 1+•, was targeted on a preparative scale by chemical oxidation. Treatment of 1 with Ag[SbF6] (1 equiv.) results in the immediate formation of an NMR-silent dark-brown solution  confirms the presence of an unpaired electron centred at g = 2.0021, which exhibits hyperfine couplings to B1 [a(11B) = 3.32 G], N1 [a(14N) = 3.14 G], N2′/N3′ [a(14N) = 2.25 G] and C3H/C4H [a(1H) = 3.18 G], hinting at the fully delocalized nature of the radical across the entire π system  is larger than for diborene radical cations (< 1.18 G)62, and in the range of mononuclear boron radical cations (2.10 − 6.43 G)59, suggesting boron radical character in 1+•.EPR analysis of tem Fig. . Interes+• can be described by several contributing resonance forms  N-centred pyridyl radical 1+•b; and (iii) NHC-centred radical 1+•c. Contributions from these three resonance structures account for (i) the majority of the unpaired spin density being located on the pyridyl and NHC heterocycles; and (ii) for NRT-derived natural bond order data for 1+• which shows reduced bond order alternation in the central pyridyl ring compared to 1 , and an exocyclic C–C bond order which is also between those determined for 1 and 12+. It is also noteworthy that in metal-free radical borylations catalyzed by 4-substituted pyridines, a pyridine-stabilized boryl radical has been invoked as the key reactive intermediate65. 1+• can be regarded as a crystalline analogue of this boryl radical species.Spin-density analysis confirms the delocalized picture, with a significant contribution on the central pyridyl ring  C-c1 with Ag[SbF6] (2 equiv.) results in successive colour changes from deep red to dark brown and ultimately to a persistent deep purple solution . X-ray crystallography confirms its identity as the target doubly oxidized dication 1+2[SbF6]2 . This value is larger than that determined for bis(carbodicarbene)[BH]2+ system (+0.36) reported by Ong et al.66, but close to that of bis(imino)[BPh]2+ dication (+1.02) reported by Inoue et al.67. As such, 12+ can also be described by several contributing resonance forms  borenium–imidazolium 12+b; or (iii) borenium dication 12+c. NICS calculations are consistent with the aromatic character of the central ring suggested by the NMR measurements (NICS(0) =  −7.55; NICS(1) = −8.99), and contrast with the non-aromatic character for 1•+ (NICS(0) = 1.52; NICS(1) = −0.87) and anti-aromatic character for 1 (NICS(0) = 6.67, NICS(1) = 4.14). Moreover, the natural bond orders obtained from NRT calculations .With regard to rms Fig. : (i) pyr1+• and 12+ sit on a crystallographic inversion centre  in the pyridyl ring, consistent with diminishing exocyclic double-bond character and concomitant aromaticity enhancement  to 1+•  → LUMO(β)) to 12+  upon successive one-electron oxidations  arising from the charge transfer from the NHB ligand to the central pyridyl ring, stretches across the visible region. This is in contrast to dications derived from wholly carbon systems which are typically restricted to absorptions in the UV region13, and hints at potential applications of all three states of this organoboron redox system in visible light photoredox catalysis.The UV–vis spectra  show a gradual red-shifting of the prominent absorption bands from 1, derived from isoelectronic NHB and NHC ligands. We demonstrate that B=N substitution confers an electronic coupling enhancement of > 1 V, enabling the isolation of a boron-containing radical cation 1+• related to Thiele’s hydrocarbon. Further single-electron oxidation affords borenium dication 12+, thereby establishing a new organoboron reversible redox triad featuring 1, 1+• and 12+. We propose that formal BN/CC substitution is critical in lowering the energy of the LUMO of the dication 12+  are too oxidizing to be experimentally accessible. As such, we perceive that our present strategy can be extended to other transient organic radicals to imbue maximal stability and facilitate their isolation.To conclude, we disclose an unsymmetrical organoboron analogue of Thiele’s hydrocarbon d6 (which was dried over potassium), with the solvent then being distilled under reduced pressure and stored under argon in Teflon valve ampoules. NMR samples were prepared under argon in 5 mm Wilmad 507-PP tubes fitted with J. Young Teflon valves. 1H, 13C{1H}, 11B{1H}, 19F{1H} NMR spectra were recorded on Bruker Avance III HD nanobay 400 MHz or Bruker Avance 500 MHz spectrometer at ambient temperature and referenced internally to residual protio-solvent (1H) or solvent (13C) resonances and are reported relative to tetramethylsilane (δ = 0 ppm). 19F resonances are referenced externally to CFCl3. Assignments were confirmed using two-dimensional 1H–1H and 13C–1H NMR correlation experiments. Chemical shifts are quoted in δ (ppm) and coupling constants in Hz. Elemental analyses were carried out by London Metropolitan University. (HCDippN)2BBr was prepared by the literature method  and AgOTf  was added CHCl3 (3 mL) and stirred for 3 days at 60 °C in an ampoule. To the suspension was added benzene (20 mL) and filtered. The filtrate was dried under vacuum to yield a solid covered in a tar-like substance. To this was added n-hexane (250 mL) and benzene (10 mL) to dislodge the tar-like substance and filtered. The filtrate was dried under vacuum to yield (HCDippN)2BOTf  as a greyish-green powder. 1H NMR : δ = 1.16 2), 1.33 2), 3.16 2), 6.01 , 7.10–7.12 , 7.18–7.22 ; 11B{1H} NMR : δ = 19.0; 19F{1H} NMR : δ = −76.68. Elemental analysis calculated for C27H36BF3N2O3S: C 60.45%, H 6.76%, N 5.22%, found: C 60.11%, H 6.55%, N 4.98%.To a mixture of (HCDippN)2BOTf  and (HCDippN)2C  in benzene (1 mL) was added pyridine  and stirred for 5 min at room temperature to form a red solution. To the solution was added K[N(SiMe3)2]  at room temperature and stirred for 5 min at room temperature to form a deep red solution. Volatiles were removed under vacuum. To the residue was added benzene (5 mL) and the mixture filtered. The filtrate was dried under vacuum to yield 1  as an orange-red powder. 1H NMR : δ = 1.18 2), 1.30 2), 1.35 2), 3.27 2), 3.38 2), 4.14 , 4.81 , 5.61 , 5.80 , 6.95 , 7.01–7.08 ; 13C{1H} NMR : δ = 23.3 (CH(CH3)2), 23.7 (CH(CH3)2), 24.5 (CH(CH3)2), 24.7 (CH(CH3)2), 28.7 (CH(CH3)2), 81.5 (Py-p-C), 107.6 (Py-CH), 117.3 (NCH), 118.5 (NCH), 121.1 (Py-CH), 123.6 (Dipp-m-CH), 123.9 (Dipp-m-CH), 127.6 (Dipp-p-CH), 128.7 (Dipp-p-CH), 132.4 (NCN), 137.7 (Dipp-i-C), 139.8 (Dipp-i-C), 146.3 (Dipp-o-C), 147.7 (Dipp-o-C); 11B{1H} NMR : δ = 19.9; UV–vis : 342 nm (ε = 16284 M−1cm−1).To a mixture of (HCDippN)2BOTf  and (HCMesN)2C  in benzene (1 mL) was added pyridine  and stirred for 5 min at room temperature to form a red solution. To the solution was added K[N(SiMe3)2]  at room temperature and stirred for 5 min at room temperature to form a deep red solution. Volatiles were removed under vacuum. To the residue was added n-hexane (25 mL) and the mixture filtered. The filtrate was dried under vacuum to yield 2  as an orange-red powder. Single crystals (yellow plates) suitable for X-ray crystallography were obtained by slow evaporation of a concentrated solution of 2 in n-pentane at room temperature. 1H NMR : δ = 1.20 2), 1.29 2), 2.07 , 2.21 , 3.32 2), 4.28 , 4.92 , 5.47 , 5.83 , 6.57 , 6.99–7.01 , 7.05–7.08 ; 13C{1H} NMR : δ = 18.4 (Mes-o-CCH3), 21.0 (Mes-p-CCH3), 23.6 (CH(CH3)2), 24.7 (CH(CH3)2), 28.7 (CH(CH3)2), 81.5 (Py-p-C), 107.6 (Py-CH), 116.3 (NCH), 118.4 (NCH), 121.4 (Py-CH), 123.6 (Dipp-m-CH), 127.6 (Dipp-p-CH), 129.3 (Mes-m-CH), 131.2 (NCN), 136.5 (Mes-p-C), 136.6 (Mes-o-C), 136.9 (Mes-i-C), 140.0 (Dipp-i-C), 146.3 (Dipp-o-C); 11B{1H} NMR : δ = 20.5; Elemental analysis calculated for C52H64BN5: C 81.12%, H 8.38%, N 9.10%, found: C 80.91%, H 8.20%, N 8.89%.To a mixture of (HCDippN)1  and AgSbF6  was added CH2Cl2 (3 mL) and stirred for 5 min at room temperature to form a dark-brown solution. The mixture was filtered and the filtrate was dried under vacuum to yield [1][SbF6]  as a dark-brown powder. Single crystals (brown rods and plates) suitable for X-ray crystallography were obtained by slow evaporation of a concentrated solution of [1][SbF6] in fluorobenzene at room temperature. X-band EPR g = 2.0021 ; UV–vis : 364 nm (ε = 13983 M−1 cm−1), 442 nm (ε = 13682 M−1cm−1); Elemental analysis calculated for C58H76BF6N5Sb: C 63.92%, H 7.03%, N 6.43%, found: C 63.77%, H 7.13%, N 6.29%.To a mixture of 1  and AgSbF6  was added CH2Cl2 (2 mL) and stirred for 5 min at room temperature to form a deep purple solution. The mixture was filtered and the filtrate was dried under vacuum to yield [1][SbF6]2  as a purple powder. Single crystals (purple rods) suitable for X-ray crystallography were obtained by slow evaporation of a concentrated solution of [1][SbF6]2 in CH2Cl2at room temperature. 1H NMR : δ = 0.85 2), 0.91 2), 1.17 2), 1.22 2), 2.21 2), 2.66 2), 6.56 , 7.14 , 7.27 , 7.38 , 7.47 , 7.69 , 7.97 , 8.03 ; 13C{1H} NMR : δ = 22.7 (CH(CH3)2), 23.4 (CH(CH3)2), 25.2 (CH(CH3)2), 25.5 (CH(CH3)2), 29.1 (CH(CH3)2), 29.9 (CH(CH3)2), 122.7 (NCH), 125.6 (Dipp-m-CH), 126.8 (Dipp-m-CH), 127.3 (Py-CH), 128.8 (Dipp-i-C), 130.1 (NCH), 130.6 (Dipp-p-CH), 133.4 (Dipp-i-C), 134.4 (Dipp-p-CH), 137.3 (Py-p-C), 137.4 (NCN), 144.7 (Dipp-o-C), 145.3 (Py-CH), 145.4 (Dipp-o-C); 11B{1H} NMR : δ = 20.1; UV–vis : 313 nm (ε = 8294 M−1 cm−1), 537 nm (ε = 5756 M−1 cm−1); Elemental analysis for C58H76BF12N5Sb2: C 52.55%, H 5.78%, N 5.28%, found: C 52.63%, H 5.51%, N 5.30%.To a mixture of Supplementary InformationPeer Review File


Asparagus cochinchinensis led to the isolation of one new furostanol saponin, named 26-O-β-d-glucopyranosyl-22α-hydroxyl-(25R)-Δ5(6)-furost-3β,26-diol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 4)]-β-d-glucopyranoside (1), along with three known congeners (2‒4). The structure of new saponin was elucidated via comprehensive inspection of its HRMS and NMR spectral data as well as chemical technology, whereas those of known ones were identified by comparison of their NMR and MS spectral data with those reported in literatures. All isolated saponins were evaluated for their cytotoxic effects on two human liver (MHCC97H) and lung adenocarcinoma (H1299) cancer cells in vitro. Among them, both 1 and 2 showed significant cytotoxicity against above mentioned cell lines. Further studies revealed that these two saponins could significantly inhibit their proliferation of MHCC97H and H1299 cells.Phytochemical investigation on the roots of The online version contains supplementary material available at 10.1007/s13659-021-00321-0. Steroid saponins, whose aglycones were usually a spirostanol or its derivatives , were coAsparagus cochinchinensis, belonging to the genus Asparagus (Liliaceae), is well-known as “Tianmendong” in China. Its roots have been historically used in Chinese folk medicine for the treatment of cough, acute and chronic bronchitis, chronic pharyngitis, hemorrhoids, and tumors for thousands of years + . It showed a positive reaction to the Ehrlich’s reagent (red color), suggesting a furostanol skeleton ‒ ion (m/z 1209.6) displayed 1 had a molecular weight (MW) of 1210.6 Da in the negative ion mode of ESI-MSn. The observed ions with m/z values of 1047.5, 901.5, and 755.4 indicated the sequential cleavage of two rhamnopyranosyl units followed by the cleavage of a glucopyranosyl moiety from the parent [M ‒ H]‒ ion (m/z 1209.6), respectively. Likewise, the MS2 spectrum also afforded m/z value of 593.4 that was indicative of the loss of one glucopyranosyl group from the C-3 position or the C-26 position , and both anomeric protons of l-rhamnoses shared α-configurations due to the chemical shifts of C-3 (δC 72.5 and 72.2) and C-5 (δC 69.3 and 68.3), respectively. In the HMBC spectrum, the long-range correlations from δH 4.88 (H-1′) to δC 77.8 (CH-3), from δH 4.73 (H-1‴″) to δC 74.9 (CH2-26), from δH 6.27 (H-1″) to δC 77.6 (CH-2′), from δH 5.74 (H-1‴) to δC 77.1 (CH-4′), and from δH 5.14 (H-1″″) to δC 84.9 (CH-4‴) established the sequence for 3-O-sugar chain as an [α-l-rhamnopyranosyl-(1 → 2)]-[β-d-glucopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 4)]-β-d-glucopyranosyl moiety and for 26-O-sugar chain as β-d-glucopyranosyl moiety, respectively. Based on the above information presented, the structure of 1 was thus elucidated to be 26-O-β-d-glucopyranosyl-22α-hydroxyl-(25R)-Δ5(6)-furost-3β,26-diol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 4)]-β-d-glucopyranoside.As for the sugar units of 2) -O-β-d-glucopyranoside (3) -β-d-glucopyranoside (1): white amorphous powder, [α] ‒46.86 ; IR (νmax): 3417, 2933, 2851, 1635, 1453, 1382, 1045 cm‒1; HRESIMS m/z 1233.5879 [M + Na]+ . 1H  and 13C  NMR spectral data, see Table 26-yranosyl- → 2- and then cultured for another 4 h. The absorbance was recorded on a microplate reader at 490 nm.The cytotoxicity of isolated compounds was determined to use the MTT method with a slight modification . Briefly5 CFU/mL. Whereafter, an inoculum of 105 CFU/mL was made to sterile 96-well plate containing tested compounds at different concentrations  at 37 ℃ for 8 h. The wells containing only broth served as growth control. The absorbance of bacterial solution was recorded on a microplate reader at 600 nm.The antimicrobial activity of isolated steroid saponins against 10 strains using the microdilution broth susceptibility assay . The strA. cochinchinensis led to the identification of one new furostanol glycoside 26-O-β-d-glucopyranosyl-22α-hydroxyl-(25R)-Δ5(6)-furost-3β,26-diol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 4)]-β-d-glucopyranoside (1) and three known one (2‒4). Meanwhile, compounds 1 and 2 exhibited cytotoxic and anti-proliferative effects on two human (MHCC97H and H1299) cancer cell lines. At the same time, compounds 1‒4 displayed moderate antimicrobial activity against C. albicans and B. subtilis, and compound 3 displayed weak antimicrobial activity against S. aureus.In summary, a chemical examination of the roots of Supplementary file1 (DOCX 6762 kb)Below is the link to the electronic supplementary material.