Chronic lymphocytic leukemia often results in secondary tumors, the most common being large B cell lymphoma known as Richter syndrome, followed by extranodal NK/T-cell lymphoma  is extremely rare.A chronic lymphocytic leukemia patient presented with nasal congestion.Nasal endoscopy identified a left nasal mass, and the pathology suggested extranodal NK/T-cell lymphoma .The patient received a course of chemotherapy.Pneumonia and coagulopathy occurred after chemotherapy, and the patient died shortly thereafter.It is essential to recognize the transformation of disease earlier in chronic lymphoblastic leukemia patient. The most common form of conversion is large B-cell lymphoma, which is called Richter syndrome. In addition, Hodgkin lymphoma, acute leukemia, multiple myeloma, and other tumors have been observed. We can distinguish whether the transformed tumor and CLL arose from the same clone by testing the mutations in the variable region of the immunoglobulin heavy chain. The factors affecting secondary tumor development include the clinical characteristics of CLL, genetics, and biology. Whether alkylating agents and nucleoside analogues increase the risk of a secondary tumor remains controversial. The prognosis is poor once CLL patients present a secondary tumor. Here, we report a rare case of CLL followed by extranodal NK/T-cell lymphoma .Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Europe and the United States,29 cells/L) during a physical examination, whereas the platelets (PLT) and hemoglobin (HB) values were normal. The patient had a fever, night sweats, and weight loss, without superficial lymphadenopathy and hepatosplenomegaly. The patient refused further diagnosis and treatment. In September 2011, the patient underwent a bone marrow puncture that showed 30.5% mature lymphocytes with 4.5% lymphoblasts. The flow cytometry suggested CLL with an abnormal B lymphocyte population accounting for 36.58% of non-erythroid cells and CD5++, CD19+, CD20+, CD23+, HLA-DR+, CD22-, CD38-, sIgMdim, and ZAP-70 expression for 87.6% of CLL cells. The patient declined treatment. In August 2012, routine laboratory results showed WBC 55.7 × 109 cells/L, lymphocyte (LY) 26.9 × 109 cells/L, PLT 69 × 109 cells/L, and HB 144 g/L. Another bone marrow puncture showed 60% mature lymphocytes with 7% lymphoblasts and with the same flow cytometry result; chromosomes: 46, XY; FISH: TP53 gene deletion. B-mode ultrasound examination found multiple enlarged lymph nodes (max 5.3 × 2.3 cm). We diagnosed the patient with CLL (Rai Staging IV). The patient was given chlorambucil , followed by 1 course of COP regimen consisting of cyclophosphamide (750 mg/m2 i.v. on day 1), vindesine (4 mg i.v. on day 1), and prednisone (60 mg/m2 i.v. daily from days 1 to 5), and 1 course of  FMD regimen consisting of fludarabine (25 mg/m2 i.v. daily from days 1 to 3), mitoxantrone (8 mg/m2 i.v. on day 1), and dexamethasone (20 mg/m2 i.v. daily from days 1 to 5). The WBC decreased to 22.1 × 109 cells/L, and LY decreased to 19.5 × 109 cells/L). Upon follow-up, the patient was in partial remission (PR). From July 2014 to July 2016, the patient underwent one course of FMD regimen consisting of fludarabine (25 mg/m2 i.v. daily from days 1 to 3), mitoxantrone (8 mg/m2 i.v. on day 1), and dexamethasone (20 mg/m2 i.v. daily from days 1 to 5); 2 courses of RFC regimen consisting of rituximab (375 mg/m2 i.v. on day 0), fludarabine (25 mg/m2 i.v. daily from days 1 to 3), and cyclophosphamide (250 mg/m2 i.v. daily from days 1 to 3); and 1 course of RFMD regimen consisting of rituximab (375 mg/m2 i.v. on day 0), fludarabine (25 mg/m2 i.v. daily from days 1 to 3), mitoxantrone (8 mg/m2 i.v. daily on day 1), and dexamethasone (20 mg/m2 i.v. daily from days 1 to 5).In 2008, a 69-year-old man presented with increased white blood cell (WBC)  regimen consisting of methotrexate (2 g/m2 i.v. on day 1), dexamethasone (40 mg i.v.i.v. daily from days 1 to 4), ifosfamide (1500 mg/m2 i.v.i.v. daily from days 2 to 4), Mesna (300 mg/m2 i.v. three times a day from days 2 to 4), etoposide (70 mg/m2 i.v. three times a day from days 2 to 4), and pegaspargase (2500 U/m2 subcutaneously on day 2). Pneumonia and coagulopathy occurred after chemotherapy, and the patient died shortly thereafter.In May 2017, a nasal endoscopy was performed and revealed a left nasal mass. The pathology suggested extranodal NK/T-cell lymphoma  infection is another factor that may be involved in the development of T-cell lymphoma in patients with CLL. We also identified Epstein-Barr virus-encoded RNA (+) cells in nasal tumor biopsies, and EBV is causally related to nasal-type T-cell lymphoma. The potential that therapy may further increase the risk of a secondary neoplasm is of great concern. Until now, no clear evidence has demonstrated that alkylating agents or purine nucleoside analogs may be associated with an increased incidence of second malignancies in patients with CLL.–10 Of course, there are many other risk factors that can lead to secondary tumors or Richter syndrome, including genetic background such as hereditable polymorphisms of BCL2 (rs4987852), CD38 (rs6449182), and low-density lipoprotein receptor-related protein 4 ; clinical features of CLL such as bulky extensive lymphadenopathy; and biological features of CLL such as the presence of NOTCH1 mutations.High-grade lymphoma of the T-cell lineage has been reported only rarely in patients with CLL.,13 Generally, the type of disease is determined based on the pathological findings followed by providing appropriate treatment. For example, diffuse large B cell lymphoma (DLBCL) can take an consist of rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone (R-CHOP) regimen, platinum-containing regimen or new drug and transplant. The choice of regimen depends on whether the 2 tumors are clonally related.,15 The case presented here was an extranodal NK/T-cell lymphoma, which was treated with a first-line SMILE regimen based on the pathologic findings, (comment NO.1), but the patient suffered from infection, coagulopathy, and respiratory failure, and died soon.There is currently no standard treatment for CLL associated with a secondary tumor or Richter syndrome.4In summary, CLL followed by extranodal NK/T-cell lymphoma  is very rare. The occurrence of this secondary tumor is associated with a variety of factors, and the efficacy of traditional treatments is poor. We hope that new drugs can provide more benefit to patients.Conceptualization: Jianping Shen.Data curation: Xiangping Wu.Investigation: Junfa Chen.Project administration: Yu Zhang.Software: Ying Chen.Writing – original draft: Hangping Ge.


Among the dietary lipids, milk fat is most complicated as it contains more than one hundred types of fatty acids and several triglycerides. Huge versatility in triglyceride and fatty composition makes it possible to convert milk fat into various fractions on the basis of melting characteristics. Functional properties of milk fat can be increased by converting it into different fractions. After cow milk, buffalo milk is the second largest source of milk and chemical characteristics of buffalo milk fat has been studied in a limited fashion. The main mandate was determination of triglyceride, fatty acid profile and antioxidant characteristics of low melting point fractions of buffalo milk fat for increased industrial applications.Buffalo milk fat (cream) was fractionated at three different temperatures i.e. 25, 15 and 10 °C by dry fractionation technique and packaged in 250 ml amber glass bottles and stored at ambient temperature for 90 days. The fraction of milk fat harvested at 25, 15 and 10 °C were declared as LMPF-25, LMPF-15 and LMPF-10. Unmodified milk fat was used as control (PBMF). Low melting point fractions were analyzed for triglyceride composition, fatty acid profile, total phenolic contents, DPPH free radicals scavenging activity, reducing power, free fatty acids, peroxide value, iodine value and conjugated dienes at 0, 45 and 90 days of storage.36, C38, C40, and C42 were 2.58, 3.68, 6.49 and 3.85% lower than PBMF. In LMPF-25, concentrations of C44, C46, C48, C50, C52 and C54 were 0.71, 1.15, 2.53, 4.8, 0.39 and 2.39% higher than PBMF. In LMPF-15, concentrations of C44, C46, C48, C50, C52 and C54 were 2.45, 4.2, 3.47, 5.92, 2.38 and 3.16% higher than PBMF. In LMPF-10, concentrations of C44, C46, C48, C50, C52 and C54 were 2.8, 5.6, 5.37, 7.81, 3.81 and 4.45% higher than PBMF. LMPF-25, LMPF-15 and LMPF-10 had higher concentration of unsaturated fatty acids as compared PBMF. Total phenolic contents of buffalo milk fat and its fractions were in the order of LMPF-10 > LMPF-15, LMPF-25 > PBMF. Storage period of 45 days had a non-significant effect on total flavonoid content. 2, 2-Diphenyl-1-picrylhydrazyl free radical scavenging activity (DPPH) free radical scavenging activity of LMP-25, LMPF-15 and LMPF-10 were 4.8, 13.11 and 25.79% higher than PBMF. Reducing power of PBMF, LMPF-25, LMPF-15 and LMPF-10 were 22.81, 28.47, 37.51 and 48.14, respectively. Estimation of free fatty acids after the 90 days of storage duration, no significant difference was found in content of free fatty acids in unmodified milk fat and low melting point fractions. Testing of peroxide value in 90 days old samples showed that peroxide value of PBMF, LMPF-25, LMPF-15 and LMPF-10 was 0.54, 0.98, 1.46 and 2.22 (MeqO2/kg), respectively. Storage period up to 45 days had a non-significant effect on anisidine value, iodine value and conjugated dienes.In LMPF-10, concentrations of CLow melting point fractions of buffalo milk fat had higher concentration of unsaturated fatty acids and more antioxidant capacity than unmodified milk fat with reasonable storage stability. Production of buffalo milk in the world is 90.3 million tons while in Pakistan average 31.252 million tons cow milk is produced. The share of buffalo milk produced in Pakistan to the world milk production is 62.8% . The lowBuffalo milk was obtained from Dairy Animals Training and Research Centre, University of Veterinary and Animal Sciences Lahore. All the chemicals used in this study were HPLC grade and obtained from Sigma Chemical Co. . For the preparation of milk fat, fat was removed from buffalo milk by cream separator. Uncultured and unsalted butter was prepared from buffalo milk, which were then converted to milk fat.oC were declared as LMPF-25, LMPF-15 and LMPF-10. Unmodified milk fat was used as control (PBMF). Each fractionation practice was repeated six times to minimize the variation [Buffalo milk fat was heated in 1-l beakers to 70 °C for 15-min in the water bath, transferred to a thermostatically controlled water bath at slowly cooled down 10 °C (1 °C/minute) held at 25, 15 and10°C for 2.5-h, low melting point fractions of buffalo milk fat were separated by vacuum filtration (600-mmHg pressure) on Buckner filtration assembly. The fraction of milk fat harvested at 25, 15 and 10ariation . The lowLow melting fractions of buffalo milk fat were characterized for free fatty acids, color, unsaponifiable matter, saponification value, iodine value, refractive index and peroxide value according to the standard methods .Following assays were performed to determine the antioxidant characteristics of LPMF, these antioxidant assays were performed at 0, 45 and 90 days of storage period.Total phenolic contents in low melting fractions of milk fat were determined according to the method prescribed by Singleton et al.  with Gal3 (2% in methanol), test tubes were incubated at room temperature of 1 h, absorbance was measured on a spectrophotometer at 420 nm, total flavonoid content were determined using Rutin as internal standard and reported as Quercetin equivalent (mg/g).Total flavonoid content of low melting fractions of milk fat was determined by the method prescribed by Nile and Khobragade . Sample DPPH free radical scavenging activity of the low melting point fractions of milk fat was estimated by the method of Sanchez-Moreno . 0.1 mM Reducing power of low melting fractions of milk fat was determined according to the method prescribed by Adesegun et al. . Brieflyα-Tocopherol in low melting point fractions of milk fat was determined by the method of Jang and Xu . 0.2 g sMilk sample (20 ml) was taken in test tube for detecting vitamin A quantification. Ammonia with concentration of 5 ml and ethanol 20 ml was added to the sample. After mixing the solution it was kept for 10 min, supernatant was extracted and BHT (0.0025%) was added to it. Rotary evaporator was used for evaporation of solvent at 35 °C. 30 ml potassium hydroxide (5% in ethanol) was added for saponification and n-hexane was used for extraction. Again, the rotary evaporator was used for evaporation. 20 μl was taken and injected into HPLC which has RP-18 column, waters 990 detector and pumping system of LC-20 AT. Mobile phase was made of Acetonitril-methanol 85:15 in isocratic system .For determination of triglyceride profile in milk, 50 mg of milk fat was taken in test tube and 1.0 mL n-hexane was added for dissolution. From this mixture, 1 μL was taken and directly injected into GC , fitted with a methyl lignoserate-coated (film thickness 0.25 l m), SP-2330  polar capillary column (RTX 65-TG) using mass spectrophotometer. The temperature of injector was 60 °C for 12 s and followed by increasing from 99 °C/min to 370 °C for holding time of 5 min. Oven temperature from 250 °C for about 2 min followed by an increase to 360 °C at 5 °C/min with holding time of 4 min. 370 °C temperature was set for detector and hydrogen was used as carrier gas with flow rate of 1.5 mL/min The split ratio was 1:  80 .Fatty acids profile was determined as fatty acid methyl esters, 300-μL melted and well mixed sample was taken into 11-mL screw capped test tube, dissolved in 3-mL isooctane and 2-mL 0.5 N sodium methoxide was added vortexed at 1500 rpm for 3-min, allowed to separate for five minutes and the supernatant was injected into GC-MS , fitted with a methyl lignoserate-coated (film thickness 0.25 l m), SP-2560 column (100 m × 0.32 mm) using flame ionization detector as per standard IUPAC method . TemperaFor the estimation of lipid oxidation in low melting point fractions of buffalo milk fat, free fatty acids, peroxide value, iodine value and anisidine values were determined . Conjugan = 3 × 3; ±SD n = 3 × 3). Data were analyzed by using two-way analysis of variance techniques to find out the effect of treatment and storage by using SAS 9.1  software. P-value of 0.05 and 0.01 was used to denote the significant and highly significant difference.Triplicate samples of each olein fraction/ storage period were taken. Every sample was analyzed three times and the data was expressed as mean (p < 0.05). Cholesterol and vitamins belong to unsaponifiable fraction, higher amount of unsaponifiable matter in the low melting point fractions can be justified by the affiliation of cholesterol and vitamins to the low melting point fractions. Nadeem et al. [Moringa oleifera oil were different from the parent oil [Chemical and physical characteristics of different fractions of buffalo milk fat are mentioned in Table m et al.  studied m et al. . Refractm et al. . Iodine m et al. . Slip merent oil . Azeem erent oil  comparedrent oil .Table 1C24-C34) in low melting point fractions of buffalo milk fat were more than unmodified milk fat. In LMPF-25, concentrations of C24, C26, C28, C30, C32 and C34 were 0.05, 0.09, 0.09, 0.08, 1.12 and 0.15% higher than PBMF. In LMPF-15, concentrations of C24, C26, C28, C30, C32 and C34 were 0.12, 0.20, 0.16, 0.16%, 1.21 and 0.30% higher than PBMF. In LMPF-10, concentrations of C24, C26, C28, C30, C32 and C34 were 0.20, 0.39, 0.30, 0.31, 0.35 and 0.84% higher than PBMF. In LMPF-25, concentrations of C36, C38, C40, and C42 were 0.23, 0.31, 2.04 and 1.03% lower than PBMF. In LMPF-15, concentrations of C36, C38, C40, and C42 were 0.98, 1.33, 5.33 and 2.33% lower than PBMF  is comprised of triglycerides present in fat globules . Several18:1 and C18:2 which were increased by 7.73, 14.26 and 28.94% at fractionation temperatures of 25, 15 and 10 °C, respectively. Foods rich in monounsaturated fatty acids are becoming more popular owing to many medical benefits and extended storage capabilities [trans form, chains having trans double bond have therapeutic activity and are bioactive [Fatty acid profile of unmodified milk fat and different fractions is mentioned in Table bilities . They albilities . The fatbilities . Functiobilities . Conjugaioactive . Earlierioactive . More thioactive . IP et aioactive  fractionioactive . Magnituioactive . Amount ioactive .Table 3FMoringa oleifera oil, Nadeem et al. [Moringa oleifera oil with soybean and sunflower oil, high oleic acid fraction of Moringa oleifera oil had more antioxidant capacity than unmodified soybean and sunflower oils. Fractionation temperature significantly affected the reducing power, the lower the fraction temperature, more was the reducing powder. Reducing power of PBMF, LMPF-25, LMPF-15 and LMPF-10 were 22.81, 28.47, 37.51 and 48.14. Reducing power of low melting point factions of milk fat reported in this investigation were higher than whole cow and buffalo milk as reported by Khan et al. [Natural antioxidants slake the dreaded free radicals which are responsible for physiological stress, ageing, cancer and atherosclerosis , 54. Scim et al.  used DPPm et al. . Fractiom et al.  comparedn et al. . Storagen et al. .Table 4ATable p > 0.05). Estimation of free fatty acids after the 90 days of storage duration, no significant difference was found in content of free fatty acids in unmodified milk fat and low melting point fractions. In oils and fats, free fatty acids are generated due to the hydrolytic activities of lipases, moisture, temperature and metal ion contamination etc. [2/kg). According to standards of European Union, allowable limit of peroxide value of oils and fats is 10 (MeqO2/kg). LMPF-10 has about 33% more C18:2 than PBMF, which may be the reason of relatively higher peroxide value in LMPF-10. Reynhout [18:2 was more than times higher than C18:1. An inverse relationship was found between peroxide value and iodine value, samples of low melting point fractions having more peroxide value showed lower iodine value. Anwar et al. [Findings on lipid oxidation of low melting point fractions of buffalo milk fat are shown in Table ion etc. . In thision etc. . From thion etc. . Free faion etc. . Among tion etc. . PeroxidReynhout  describer et al.  establis44 to C54 were intensified in the low melting point factions. Low melting point fractions harvested at 15 and 10oC had stronger free radical scavenging capacity than unmodified milk fat. α-tocopherol and vitamin A were also intensified in low melting point fractions. Up to 45 days, peroxide value, anisidine value, iodine value and conjugated dienes in all the three low melting point fractions were not different from unmodified milk fat. Low melting point fractions can be extracted from milk fat for the formulation of functional foods and industrial applications.Low melting point fractions of buffalo milk fat had higher amount of unsaturated fatty acids and conjugated linoleic acids than unmodified milk fat. Triglycerides from C


LVS-019 with cyanopyridine scaffold, showed good IDO1 inhibitory activity. To discover more compounds with similar structures to LVS-019, a shape-based model was then generated on the basis of it and the second-round virtual screening was carried out leading to 23 derivatives. Molecular docking studies suggested a possible binding mode of LVS-019, which provides a good starting point for the development of cyanopyridine scaffold compounds as potent IDO1 inhibitor. To improve potency of these hits, we further designed and synthesised another 14 derivatives of LVS-019. Among these compounds, LBJ-10 showed improved potency compared to the hits and displayed comparable potency to the control GDC-0919 analogue. LBJ-10 can serve as ideal leads for further modifications as IDO1 inhibitors for cancer treatment.With the aim of discovering novel IDO1 inhibitors, a combined similarity search and molecular docking approach was employed to the discovery of 32 hit compounds. Testing the screened hit compounds has led to several novel submicromolar inhibitors. Especially for compounds  L-tryptophan conversion to N-kynurenine along the kynurenine pathway,L-Trp, D-Trp, and tryptamine, and is ubiquitously expressed in many tissues,D-tryptophan  which currently is in phase I clinical trials in subjects with recurrent advanced solid tumours,3), which was in phase II/III clinical trials, used as a monotherapy as well as in combination use with various antibodies, for the treatment of advanced or metastatic cancers4), which is under phase I clinical trials for the treatment of patients with grade IV glioblastoma or grade III anaplastic glioma,5), which is in phase I/II clinical trials in combination with nivolumab in patients with advanced cancersSHR-9146  with favourable preclinical oral bioavailability and safety profiles, which is under phase I clinical trials in subjects with advanced solid tumours. A novel prodrug of indoximod (named NLG-802 or NCT03164603) with enhanced pharmacokinetic properties was also entered into Phase I study recently by NewLink Genetics. The structures of SHR-9146 and NLG-802 have not been disclosed until now.Indoleamine 2,3-dioxygenase 1 (IDO1) is a haeme-containing enzyme that catalyses the first and rate limiting step of 6) of currently most promising IDO1 inhibitor INCB024360 was discovered through high-throughput screening (HTS) of Incyte’s corporate collection,LVS-19 attracted us the most. We then selected LVS-19 to generate the rapid overlay of chemical structures (ROCS) model. With strictly chemical similarity analysis, a second-round shape-based screening was performed. Twenty-three derivatives LVS-33∼LVS-56 were finally retained and purchased from Specs database. They were also evaluated for their IDO1 inhibitory activity. A schematic of the overall VS protocol is presented in LBJ-01∼LBJ-14) of LVS-019 were designed and synthesised through rational drug designation.With only these IDO1 inhibitors under clinical trials and no drug has been approved, discovering novel IDO1 inhibitors suitable for development into clinical candidates is still in great demand. Further exploring these IDO1 inhibitors, we found that many of them with new structural scaffolds were discovered by structure-based design, high-throughput screening, and natural product derivatisationwww.specs.net) and these compounds were prepared with the module of Search Conformations in MOE2016.09The Specs database file (version 01/2012) was downloaded from the official Web site (LVS-019. The parameters for the ROCS run were set as follows: rankby = combo and besthits =1. The screening score for a particular database compounds was set to the maximum combo score between the database compound and model compounds.The shape-based pharmacophore model in VS was generated by employing ROCSSupporting Information.) These ligands were docked back into their corresponding protein structures using Gold5.1 (CCDCs software GOLD version 5.1),The crystallographic coordinates of human IDO1-inhibitors  were used to conduct native-dockingThe complexes used in native-docking were also selected to perform cross-docking evaluation. After superposing the crystallographic complexes, the native ligands were extracted, merged in a unique database and docked into each protein structure with five docking combination protocols used in native-docking. For each of the five docking protocols and six PDB structures, a database of five ligands was docked to each of the six protein structures (the self-ligand was excluded for the cross-docking computation to follow a strict cross-docking procedure). Ten poses were generated for each of the ligand–protein couples and the RMSD values between the top-scoring pose and the crystallographic conformation of the ligand were computed. The evaluation of each protein-docking protocol couple was performed considering the RMSD of the top-scoring poses of each of the five native ligands docked on the same protein with the same protocol.GOLD was a powerful genetic algorithm (GA) method for conformation search and docking. In the present study, the binding site was defined by carefully analysing the protein–ligand interactions between IDO1 and its inhibitor. Residues around the original ligand (radius 8.0 Å) were also included in the active site. All water molecules have been removed from the protein structure. Docking studies were performed using the standard default settings with 10 GA runs on each molecule. For each of the GA runs, a maximum of 125,000 operations were performed. The binding energy of the ligands with protein was predicted using the Goldscore by choosing in the appropriate scoring function goldscore.p450_pdb.params file from those that have been supplied with the GOLD installationN-formylkynurenine to kynurenine, 10 µL of NaOH (1M) was added and incubated at 60 °C for 30 min. Then the mixture was cooled at 0 °C and centrifuged at 10,000 rpm for 10 min. Lastly, 100 µL supernatant form each well was transferred to a new 96-well plate and mixed at equal volume with 2% (w/v) p-dimethylaminobenzaldehyde  in acetic acid. The yellow colour generated from the reaction with kynurenine was measured at 480 nm. The percentage of inhibition is reported as (100 − (A/B × 100))/100, where A is the IDO1 activity in the presence of the test compound, and B is the IDO1 activity in the absence of the test compound.The recombinant human IDO1 was expressed and purified as previously reported4 per well. On the next day, human IFN-γ (20 ng/well) and compounds in a total volume of 200 µL culture medium containing 15 µg/mL of L-tryptophan were added to the cells. After incubation for 24 h, 140 µL of supernatant was mixed with 10 µL of 6.1 N trichloroacetic acid and the mixture was incubated for 30 min at 60 °C. The reaction mixture was then centrifuged for 20 min at 4000 rpm at 0 °C to remove sediments. About 100 µL of the supernatant was mixed with 100 µL of 2% (w/v) p-DMAB in acetic acid and measured at 480 nm. The final DMSO concentration was 0.5%. The data were analysed using Graphpad Prism 6.HeLa cells were seeded in 96-well culture plates at a density of 5 × 10LBJ-10 (20 µM in 0.5% DMSO) to IDO1 (5 µM) in phosphate buffer (pH 6.5) using a Tecan Safire2 microplate reader.Absorbance spectra (250–480 nm) were measured immediately after addition of compound LBJ-10 solution was added into the mixture. The reaction, at 37 °C, was started by the addition of the substrate and terminated after 60 min by adding 200 µL of 30% (w/v) trichloroacetic acid and further incubated at 50 °C for 30 min to hydrolyse N-formylkynurenine to produce kynurenine. After centrifugation at 12,000 rpm for 15 min at 20 °C, kynurenine in the supernatant (1 mL) was measured by a HPLC system with a reversed phase column (4.6 mm × 15 cm) of GL Sciences Inc. The mobile phase was 10 mM ammonium acetate containing 10% (w/v) methanol, and kynurenine was detected by absorbance at 360 nm. All determinations were carried out in duplicate. The data presented are average values. Based on the results of the activity assay, a function of rate ([V]) against enzyme amount ([E]) was proposed.The standard assay mixture (1 ml) contained 50 mM potassium phosphate buffer (pH = 6.5), 25 mM neutralised ascorbic acid, 10 µM methylene blue, 100 µg/mL catalase, 50 µM L-Trp, DMSO (0.5%), and eight different concentrations of IDO1. In addition, 1 µM ,The aqueous solubility was determined on a Gemini Profiler instrument (pION) by the “goldstandard” Avdeef–Bucher potentiometric titration method2. MTT (50 µg) was added and the cells were incubated for another 4 h. Medium/MTT solutions were removed carefully by aspiration, the MTT formazan crystals were dissolved in 100 µL of DMSO, and absorbance was determined on a plate reader at 560 nm.Growth inhibition was determined by the MTT colorimetric assay. HeLa cells were plated in 96-well plates at a density of 10,000 cells/mL and allowed to attach overnight (16 h). Cells were plated in 96-well plates at a density of 10,000 cells/mL and allowed to attach overnight (16 h). Cells were then exposure to 50 µM of test compounds in triplicate for 48 h at 37 °C under a humidified atmosphere containing 5% CO1H NMR and 13C NMR spectra were recorded on a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane as internal standard. Chemical shifts are given in ppm (δ). The multiplicities are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartette; m, multiplet; br s, broad singlet. IR spectra were recorded on a Nicolet iS10 Avatar FT-IR spectrometer using KBr film. ESI-mass and high-resolution mass spectra (HRMS) were recorded on a Water Q-Tofmicro mass spectrometer. Analytical results are within 0.40% of the theoretical values. Purity of the compounds was analysed by HPLC (Agilent Technologies 1260 Infinity) using 80:20 MeOH/H2O as the mobile phase with a flow rate of 0.8 ml/min on a C18 column. All tested compounds exhibited greater than 95% purity unless otherwise noted.All chemicals and reagents were purchased from commercial sources. Organic solutions were concentrated in a rotary evaporator (Bu¨chi Rotavapor) below 55 °C under reduced pressure. Silica gel thin-layer chromatography was performed on precoated plates GF-254 and visualised under UV light. Melting points were determined with a Melt-Temp II apparatus. m/z [M + H]+ calculated for C6H8F3O2: 169.0471, found: 169.0477.To the ethoxyethene  in pyridine  was added trifluoroacetic anhydride by dripping slowly at 0 °C. Then stirring for 12 h at room temperature. The mixture was adjusted to neutral with 2 N HCl. The organic was washed with water and brine successively, dried over anhydrous sodium sulphate, and concentrated in vacuum to obtain yellow oily liquid . HRMS 16  was dissolved in 50 ml of ethanol, and nitrile acetamide  was added at 0 °C. Then sodium ethoxide  was slowly added. The mixture was stirred under reflux for 12 h and acidified with 2 N HCl to pH =3–4. Subsequently, the solvent was concentrated in vacuum and diluted with ethyl acetate. The solution was washed with water and brine, dried over anhydrous sodium sulphate, and concentrated in vacuum to obtained the product as a yellow solid . HRMS m/z [M + H]+ calculated for C7H4F3N2O: 189.0270, found: 189.0270.Intermediate 17  was added to the bottle, and phosphorus oxychloride  was added. The mixture was refluxed for 2.5 h. Removing excess phosphorus oxychloride under reduced pressure, then poured the residue to the ice water and washed with sodium bicarbonate. The mixture was then extracted twice with AcOEt, washed five times with a saturated solution of NaCl, dried over Na2SO4 and concentrated. The crude product was purified by flash column chromatograph on silica gel (PE/EA =10:1) to give 18a in 45% yield as a milk white powder. HRMS m/z [M + H]+ calculated for C7H3ClF3N2: 206.9931, found: 206.9933.Compound 21a . HRMS m/z [M + H]+ calculated for C8H9FNO2: 170.0612, found: 170.0614.To a solution of 4-fluoroaniline  at room temperature was added glycolic acid . The mixture was then stirred at 115 °C for 5.5 h and quenched at room temperature by addition of a saturated solution of sodium bicarbonate then obtain precipitate 21 b . HRMS m/z [M + H]+ calculated for C8H9FNOS: 186.0383, found: 186.0382.To a solution of 4-fluoroaniline  at room temperature was added mercaptoacetic acid . The mixture was then stirred at 125 °C for 6 h and quenched at room temperature by addition of a saturated solution of sodium bicarbonate then obtain precipitate 21c . HRMS m/z [M + H]+ calculated for C8H10NOS: 168.0478, found: 168.0472.To a solution aniline  at room temperature was added mercaptoacetic acid . The mixture was then stirred at 110 °C for 6 h and quenched at room temperature by addition of a saturated solution of sodium bicarbonate then obtain precipitate 21d . HRMS m/z [M + H]+ calculated for C8H9FNOS: 186.0383, found: 186.0386.To a solution 2-fluoroaniline  at room temperature was added mercaptoacetic acid . The mixture was then stirred at 120 °C for 6 h and quenched at room temperature by addition of a saturated solution of sodium bicarbonate then obtain precipitate 21e . HRMS m/z [M + H]+ calculated for C8H8ClFNOS: 219.9994, found: 220.0000.To a solution 3-chloro-4-fluoroaniline  at room temperature was added mercaptoacetic acid . The mixture was then stirred at 150 °C for 6 h and quenched at room temperature by addition of a saturated solution of sodium bicarbonate then obtain precipitate 21f . HRMS m/z [M + H]+ calculated for C9H12NOS: 182.0634, found: 182.0636.To a solution 4-methylaniline  at room temperature was added mercaptoacetic acid . The mixture was then stirred at 105 °C for 6 h and quenched at room temperature by addition of a saturated solution of sodium bicarbonate then obtain precipitate 21 g . HRMS m/z [M + H]+ calculated for C9H12NO2S: 198.0583, found: 198.0586.To a solution 4-methoxyaniline  at room temperature was added mercaptoacetic acid . The mixture was then stirred at 100 °C for 6 h and quenched at room temperature by addition of a saturated solution of sodium bicarbonate then obtain precipitate 21a  in anhydrous DMF at room temperature was added K2CO3 . The mixture was then stirred at 20 °C for 15 min, the reaction was quenched with water then the product was obtained by filtration and the precipitate was purified by silica gel chromatography  to give the title product as a white solid . m.p. 234–236 °C. HRMS m/z [M + Na]+ calculated for C14H10FN3OSNa: 294.0655, found: 294.0659. 1H NMR  δ: 10.27 , 8.42 , 8.33 , 7.58 , 7.24–7.12 , 5.09  ppm. 13C NMR  δ: 165.6, 162.5, 158.0 (1JCF = 240.0 Hz), 151.5, 144.2, 134.9, 134.8, 120.9 (3JCF = 8.3 Hz), 117.8, 115.4 (2JCF = 22.5 Hz), 95.7, 64.7 ppm.To a solution of 2-chloro-3-nitrile pyridine  and 21b following the synthesis route of LBJ-01. Yellow solid . m.p. 215–217 °C. HRMS m/z [M + H]+ calculated for C15H12FN2OS: 287.0654, found: 287.0668. 1H NMR  δ: 9.44 , 8.10 , 7.89 , 7.73–7.68 , 7.51 , 7.42 , 7.29 , 7.16  ppm. 13C NMR  δ: 164.9, 162.5, 158.0 (1JCF = 238.6 Hz), 148.0, 144.3, 135.2 121.0 (3JCF = 7.5 Hz), 116.5, 116.4, 115.2 (2JCF = 21.8 Hz), 114.5, 109.6, 35.1 ppm.The title compound was prepared from 2-chlorobenzonitrile and 21b following the synthesis route of LBJ-01. Yellow solid . m.p. 198–201 °C. HRMS m/z [M + H]+ calculated for C14H11FN3OS: 288.0601, found: 288.0605. 1H NMR  δ: 10.41 , 8.71–8.69 , 8.29–8.26 , 7.63–7.58 , 7.38–7.33 , 7.17 , 4.26  ppm.The title compound was prepared from 2-chloro-3-cyanopyridine and 21b following the synthesis route of LBJ-01. Yellow solid . m.p. 206–209 °C. HRMS m/z [M + H]+ calculated for C14H11FN3OS: 288.0601, found: 288.0603. 1H NMR  δ: 10.37 , 8.69–8.67 , 8.27–8.24 , 7.61–7.56 , 7.36–7.31 , 7.18–7.12 , 4.24  ppm.The title compound was prepared from 3-chloro-4-cyanopyridine and 21b following the synthesis route of LBJ-01. White solid . m.p. 232–234 °C. HRMS m/z [M + H]+ calculated for C14H11FN3OS: 288.0601, found: 288.0603. 1H NMR  δ: 10.48 , 8.86 , 8.67 , 7.62–7.56 , 7.21–7.13 , 4.23  ppm.The title compound was prepared from 4-chloro-3-cyanopyridine and 21b following the synthesis route of LBJ-01. Yellow solid . m.p. 211–213 °C. HRMS m/z [M + H]+ calculated for C14H11FN3OS: 288.0601, found: 288.0605. 1H NMR  δ: 9.63 , 8.70–8.68 , 8.46–8.43 , 7.74–7.69 , 7.57–7.53 , 7.18 , 6.94  ppm. 13C NMR  δ: 163.5, 158.0 (1JCF = 240.0 Hz), 147.1, 146.8, 146.6, 135.1, 132.2, 131.7, 123.1 (3JCF = 7.5 Hz), 122.5, 115.0 (2JCF = 22.5 Hz), 42.8 ppm.The title compound was prepared from 3-chloro-2-cyanopyridine and 21b following the synthesis route of LBJ-01. Yellow solid . m.p. 238–240 °C. HRMS m/z [M + H]+ calculated for C15H13FN3OS: 302.0758, found: 302.0758. 1H NMR  δ: 10.39 , 8.11 , 7.61–7.57 , 7.20–7.13 , 4.17 , 2.5  ppm.The title compound was prepared from 2-chloro-3-cyanopyridine and 18a and 21b following the synthesis route of LBJ-01. Yellow solid . m.p. 201–204 °C. HRMS m/z [M + H]+ calculated for C15H10F4N3OS: 356.0475, found: 356.0477. 1H NMR  δ: 10.42 , 8.56 , 7.82 , 7.58–7.53 , 7.15 , 4.24  ppm. 13C NMR  δ: 164.0, 158.6 (1JCF = 238.5 Hz), 149.0, 137.4, 135.4, 132.0, 128.0, 123.9, 122.9 (3JCF = 7.5 Hz), 122.6, 114.9 (2JCF = 22.5 Hz), 97.47, 30.65 ppm.The title compound was prepared from 18a and 21d following the synthesis route of LBJ-01. White solid . m.p. 208–211 °C. HRMS m/z [M + H]+ calculated for C15H10F4N3OS: 356.0475, found: 356.0473. 1H NMR  δ: 10.14 , 8.56 , 7.83 , 7.30–7.23 , 7.16–7.212 , 4.33  ppm.The title compound was prepared from 18a and 21e following the synthesis route of LBJ-01. White solid . m.p. 227–229 °C. HRMS m/z [M + H]+ calculated for C15H9ClF4N3OS: 390.0085, found: 390.0077. 1H NMR  δ: 10.58 , 8.56 , 7.87–7.81 , 7.46–7.35 , 4.25  ppm. 13C NMR  δ: 163.6, 158.4 (1JCF = 238.5 Hz), 147.5, 145.5, 142.7, 137.5, 135.0, 133.5, 123.3 (3JCF = 8.0 Hz), 116.4, 115.1 (2JCF = 22.0 Hz), 101.5, 54.25 ppm.The title compound was prepared from 18a and 21c following the synthesis route of LBJ-01. Yellow solid . m.p. 230–233 °C. HRMS m/z [M + H]+ calculated for C15H11F3N3OS: 338.0569, found: 338.0579. 1H NMR  δ: 10.34 , 8.55 , 7.81 , 7.54 , 7.30 , 7.05 , 4.25  ppm. 13C NMR  δ: 164.90, 162.54, 144.32, 138.84, 128.65, 123.38, 119.24, 116.49, 116.45, 116.41, 114.54, 35.25 ppm.The title compound was prepared from 18a and 21f following the synthesis route of LBJ-01. Yellow solid . m.p. 212–214 °C. HRMS m/z [M + H]+ calculated for C16H13F3N3OS: 352.0726, found: 352.0733. 1H NMR  δ: 10.24 , 8.57–8.54 , 7.81 , 7.41 , 7.10 , 4.24 , 2.25  ppm. 13C NMR  δ: 164.63, 162.57, 148.03, 144.29, 136.33, 132.31, 129.02, 119.26, 116.42, 116.38, 116.34, 114.53, 109.63, 102.88, 35.22, 20.38 ppm.The title compound was prepared from 18a and 21g following the synthesis route of LBJ-01. Yellow solid . m.p. 217–219 °C. HRMS m/z [M + H]+ calculated for C16H13F3N3O2S: 368.0675, found: 368.0681. 1H NMR  δ: 10.20 , 8.55 , 7.82 , 7.46–7.44 , 6.89–6.86 , 4.22 , 3.72  ppm. 13C NMR  δ: 164.38, 155.34, 144.29, 131.97, 120.83, 116.45, 116.38, 114.54, 113.81, 55.12, 54.78, 35.13, 27.31 ppm.The title compound was prepared from 21e following the synthesis route of LBJ-01. White solid . m.p. 86–88 °C. HRMS m/z [M-H]– calculated for C14H8ClF4N2OS 363.0023, found 363.0017; 1H NMR  δ: 10.50 , 7.95–7.86 , 7.70 , 7.59 , 7.45–7.37 , 4.11  ppm.The title compound was prepared from 2-chloro-6-(trifluoromethyl)pyridine and ,ROCS is a fast shape comparison application which developed based on the principle that molecules have similar shape if their volumes overlay well and any volume mismatch is a measure of dissimilarityThe combination of shape matching (shape Tanimoto) and functional group matching (colour Tanimoto) expressed as Tanimoto combo score. The 3D similarity was ranked by the Tanimoto ComboScore method. Both shape and colour score vary from 0 to 1 (besthits = 1), the combo score as the sum varies between 0 and 2In this study, three representative compounds with diverse scaffolds, GDC-0919, INCB024360, and Amg-1, were selected as the reference molecule to generate the ROCS models. The 3D conformers of GDC-0919 and Amg-1 were obtained through the crystal structures . As for INCB024360, a low energy 3D conformer was generated using the MMFF94x forcefield (MOE 2016.08). The molecular shapes of these three compounds were depicted in yellow shadow . Then, t8, PDB code 2D0T)9, PDB code 4PK5)10 (PDB code 5EK2), 11 (PDB code 5EK3), 12 (PDB code 5EK4), and 13 (PDB code 5ETW)Up to date, nine crystal structures of IDO1 have been deposited in the RSCB Protein Data Bank (PDB). Among them, six structures with crystallographic resolutions higher than 3.0 Å were utilised in this work. These structures are all IDO1 with its potent inhibitors, including 4-phenylimidazole , CDOCKET , Alpha PMI , AutoDock , and Glide , AutoDock, and Glide, we first performed the re-docking experiments. As shown in 2+ ion of haeme. In addition, as shown in the interaction between the native ligand and IDO1, the formed hydrophobic interactions with the hydrophobic resides Try126, Cys129, and Phe163 of IDO1 could improve the binding affinity. Top 10% of the molecules were recruited and further visually examined. In the process of visual inspection, we removed the reported compounds against IDO1 and ruled out the structures with the unreasonable binding modes discreetly . Finally, 32 compounds were purchased form Specs for in vitro testing . Among these compounds LVS-019 was discovered as the most potent IDO1 inhibitors.After the previous shape-based VS, the remaining compounds were submitted docking simulation using the genetic optimisation for ligand docking (Gold5.1) software package targeting multi-crystal IDO1 structures. The binding energy of the ligands with protein was predicted using GOLD score implemented in GOLD. The total GOLD score, which is represented as “Fitness,” was calculated from the contribution of hydrogen bonds and van der Waals interactions between protein and ligand as well as the contribution of intramolecular hydrogen bonds and intramolecular strain in the ligand. The binding site was defined as being any volume within 8 Å of the ligand in the crystal pose, and the residues Arg231, Leu384, Phe226, Gly262, Ser263, Ala264, Phe163, Ser167, Tyr126, Cys129, and His346 were found to be interacting residues as shown in LVS-019 was performed. About 500 Compounds with similar molecular shape of LVS-019 were screened out. The structural similarity of these molecules was then analysed using Functional-Class Fingerprints_6 (FCFP_6) molecular fingerprintLVS-019. After such screening, 48 derivatives of LVS-019 were retained and processed to dock by Gold5.1 to rule out the structures with the unreasonable binding modes. Finally, 23 commercial available derivatives of LVS-019 were retained and purchased to bioevaluation .As we knew, it is equivocal to determine the activity of a chemical scaffold judging by one compound due to the potential experimental error. In order to avoid the deviation in the subsequent molecular design and confirm the activity of screening hits, a further chemical similarity search based on 55. To ensure that our compounds did not interfere with biological assays, representative compounds and water  with the addition of different concentrations of kynurenine in the absence of IDO1 protein. After p-dimethylaminobenzaldehyde (p-DMAB) was added, the kynurenine concentrations were determined by measuring the absorbance at OD 480 nm. The results showed that both of the test compounds did not interfere with the signal detection. Initial enzyme-based IDO1 inhibitory activity assay was conducted for 32 compounds obtained from VS at concentrations of 10 µM using L-1-MT and GDC-0919 analogue (racemic mixture of GDC-0919) as a positive control. The results are reported in Table S1. Based on the assay, five compounds  revealed potential inhibitory activity to IDO1 with inhibition rates more than 50% at 10 µM. Then, we measured the dose response behaviours of these compounds, the IC50 values of these compounds are shown in LVS-019 with cyanopyridine scaffolds had the highest activity with IC50 value of 2.64 µM. Based on the promising scaffold of LVS-019, further 23 derivatives were discovered and evaluated. The structures and the IC50 values of these 23 derivatives are shown in Figures S2 and S4, and Table S2. Among these derivatives, several compounds  showed similar potency compared with LVS-019  were also assayed for their ability to inhibit tryptophan degradation and kynurenine production in the HeLa kynurenine assay. LVS-019, LVS-039, LVS-048, and LVS-052. The Hill slope of these dose response curves did not deviate from 1, indicated that these compounds may be specific inhibitors of IDO1. These four compounds displayed cellular IDO1 inhibitory activity in the HeLa cell line with EC50 value in the range of 1.64–9.40 µM. More importantly, a good correlation between the recombinant enzyme-based and cellular-based assays confirmed that these cyanopyridine scaffold compounds can be developed to potent IDO1 inhibitors. In addition, since the inhibition of tryptophan degradation could simply be an effect of cytotoxicity, thus the determination of cell viability was necessary when reporting cellular IC50 values. In the initial work, all of the screened compounds were determined for their cell viability using the classical MTT assy. The results are shown in Figures S5 and S6, most of these compounds displayed negligible level of toxicity against HeLa cells at 50 µM.The HeLa-based cellular assay is informative for drug development, as it evaluates not only the IDO1 inhibitory effect of the compounds but also their capacity to permeate the cell, their inhibition of tryptophan and kynurenine transport and their potential cytotoxicityLVS-019, LVS-039, LVS-048, and LVS-052 to the top-scored protein structures (PDB code: 4PK5). The general molecular orientation and the spatial location of the chemical features of these inhibitors were similar to that of native ligand Amg-1. The nitrile group in pyrimidine ring of these compounds directly binds to the iron-haeme and pyrimidine ring is sitting at the pocket A. Furthermore, it is predicted that the ring B occupied the pocket B which is composed of residues Arg231, Phe262, and Leu234. More importantly, the ring B of these compounds could form π–π interaction with the Phe226. These four potent compounds were more effective than the other compounds, supported that the halogen or nitrile substitution at the B ring could form an additional electrostatic interaction of Arg231 which is necessary for improving the IDO1 inhibitory activity.LVS-019 derivatives (LBJ-01∼LBJ-13) with simplified structures and discussed the preliminary structure–activity relationship (SAR) of these series of compounds to find promising lead compound. In addition, to investigate the importance of the cyano group for IDO1 binding, we also designed and synthesised the compound LBJ-14 which lacks the group.Though these four screened compounds have been confirmed that possess potential IDO1 inhibitory activity and stable binding properties. However, there are still several defects for these compounds being promising lead compounds to further drug discovery, such as the moderate IDO1 inhibitory activity at micromole level and low water solubility (18 µg/mL). Thus, we designed several novel LBJ-01∼LBJ-13 are shown in 17 was synthesised starting from ethyl vinyl ether 15 in three steps by a modification of the previously report procedure15 followed by condensation with cyanoacetamide provided pyridine 17, which was subsequently converted to 2-chloropyridine by POCl3. Intermediate 21 was prepared through the neat acylation of substituted phenylamine (20) with glycolic acid (19a) or mercaptoacetic acid (19b). Finally, the target compounds (LBJ-01∼LBJ-13) were obtained by nucleophilic aromatic substitution using the intermediates 17a–17b with 20 under weak base condition.The synthesis routes used to prepare derivatives LBJ-08 (IC50 = 1.87 ± 0.83 µM) and LBJ-10 (IC50 = 0.84 ± 0.05 µM) showed improved IDO1 inhibitory activity than LVS-019 (IC50 = 2.64 ± 0.94 µM) . The SAR studies demonstrated that the remove of the isopropyl group (LBJ-03) would reduce IDO1 inhibitory activity. LBJ-1 with oxygen atom at the linker showed worse IDO1 activity compared with LBJ-3, indicated that the sulphur atom at the linker also has positive effect for the IDO1 inhibitory activity. Changing the 3-cyanopyridine scaffold (LBJ-03) to benzene (LBJ-02) or other substituted pyridine with nitrile group at different positions (LBJ-04∼LBJ-06) failed to show stronger inhibition efficiency. The 5-trifluoromethyl substituted cyanopyridine compound (LBJ-08) showed stronger IDO1 inhibitory activity than the unsubstituted compound LBJ-03. Based on LBJ-08, further modification focussed on the B ring, a series of different anilines substituted compounds (LBJ-09∼LBJ-13) was synthesised to explore the SAR, and most of the assayed compounds were less potent than LBJ-08 except for LBJ-10 which showed improved potency to that of LBJ-08. Compound LBJ-14 was much weaker than compound LBJ-10, indicated the necessity of the presence of cyano group in these series of compounds. With IDO1 inhibitory activity at the nanomole level, compound LBJ-10 could be a promising IDO1 inhibitor for further study. We also determined the solubility of these compounds using the “goldstandard” Avdeef–Bucher potentiometric titration method. To our disappointment, except for compound LBJ-03 with four times more soluble (75 µg/mL) than LVS-019, other compounds were on the same level with it. Thus, we would further focus on improving the drug-like properties of these series of compounds in the near future.These 14 synthesised molecules were evaluated for their IDO1 inhibitory activity using an enzyme-based method. Encouragingly, compound LBJ-10  against the enzyme amount ([E])  58,59. Thnhibitor . More spLVS-019 with cyanopyridine scaffold, showed the best IDO1 inhibitory activity. To discover more compounds with similar structures to LVS-019, a ROCS model was then generated on the basis of LVS-019 and then the second-round VS was carried out. Then the hits obtained by similarity search were further evaluated by GOLD, and 23 compounds were purchased to test in vitro leading to the discovery of another three potent compounds . To improve potency of these hit compounds, we designed and synthesised another 14 derivatives of LVS-019 through rational drug designation. As we expected, compound LBJ-10 showed improvement potency compared to the hit compound and displayed comparable potency to the control GDC-0919 analogue. In this direction, the in silico approach described can also be used to screen existing database to identify derivatives with desired activity. Further studies on the molecular optimisation of the other hits to provide some other potent IDO1 inhibitors with novel scaffolds are currently underway.With the combined strategy of similarity search and molecular docking study, a diverse range of scaffolds of IDO1 inhibitors were identified from the Specs database. The shape-based screening model was developed using the ROCS method, based on the reported representative IDO1 inhibitors INCB024360, GDC-0919, and Amg-1. Subsequently, optimal docking process was further employed to screen the compounds and 32 hit compounds were finally discovered. Testing the screened hit compounds has led to several submicromolar inhibitors with novel scaffolds. Especially for compounds


The CFO powders were prepared as a reference via a typical solid solution process. In particular, two types of heterogeneous CZFO powders were prepared via a stepwise solid solution process. Porous-CFO and dense-CFO powders were synthesized by calcination in a box furnace without and with pelletizing, respectively. Then, heterogeneous structures of pCZFO and dCZFO powders were prepared by Zn-substitution on calcined powders of porous-CFO and dense-CFO, respectively. Compared to the CFO powders, the heterogeneous pCZFO and dCZFO powders exhibited maximal magnetic susceptibilities (χmax) at lower Hdc values below ±50 Oe and ±10 Oe, respectively. The Zn substitution effect on the Hdc shift was more dominant in dCZFO than in pCZFO. This might be because the Zn ion could not diffuse into the dense-CFO powder, resulting in a more heterogeneous structure inducing an effective exchange-spring effect. As a result, ME composites consisting of 0.948Na0.5K0.5NbO3–0.052LiSbO3 (NKNLS) with CFO, pCZFO, and dCZFO were found to exhibit Hopt. = 966 Oe (NKNLS-CFO), Hopt. = 689–828 Oe (NKNLS-pCZFO), and Hopt. = 458–481 Oe (NKNLS-dCZFO), respectively. The low values of Hopt. below 500 Oe indicate that the structure of magnetostrictive materials should be considered in order to obtain a minimal Hopt. for high feasibility of ME composites.In this study, magnetostrictive powders of CoFe Since the year 2000, magnetoelectric (ME) response has been a topic of interest in the development of energy-harvesters, sensitive magnetic sensors, and magnetically driven memories, or magnetoelectric transducers ,2,3. TheHopt.) on the order of over several thousand Oersteds (Oe), which is a serious drawback limiting practical ME applications [αME) was obtained at high values of Hopt. above 1000 Oe from various compositions of Pb(Zr0.52Ti0.48)O3-Ni0.8Zn0.2Fe2O4 (αME = 54.4 mV/cm·Oe at Hopt. = 1000 Oe), BaTiO3-Co0.6Zn0.4Fe1.7Mn0.3O4 (αME = 73 mV/cm·Oe at Hopt. > 2000 Oe), BaTiO3-CoFe2O4 (αME = 17.04 mV/cm·Oe at Hopt. > 15000 Oe), Ba0.85Ca0.15Ti0.9Zr0.1O3-CoFe2O4 (αME = 1.028 mV/cm·Oe at Hopt. > 8000 Oe), and Na0.5Bi0.5TiO3-CoFe2O4 (αME = 0.42 mV/cm·Oe at Hopt. >2500 Oe) [Hopt. values of 500–1000 Oe were reported when investigating the size effect of magnetostrictive particles in BaTiO3-NiFe1.98O4 (αME = ~252 mV/cm·Oe at Hopt. = 500–1000 Oe), the sintering temperature effect in Pb(Zr0.52Ti0.48)O3-NiCo0.02Cu0.02Mn0.1Fe1.8O4 (αME = 63 mV/cm·Oe at Hopt. = 600 Oe), and the piezoelectric phase effect in Pb(Zr0.52Ti0.48)O3-Nix1−ZnxFe2O4 (αME = 190 mV/cm·Oe at Hopt. = 800 Oe), there is still a need to decrease Hopt. below 100 Oe for a high feasibility of particulate ME composite [However, reliable ME voltage from 3-0 type particulate composites can only be obtained under an optimal dc magnetic field  ,13,14,15omposite ,17,18.Hopt. values in a particulate ME composite system. In particular, magnetostrictive powders of CoFe2O4 (CFO), Zn-substituted porous-CFO (pCZFO) and Zn-substituted dense-CFO (dCZFO) were respectively prepared to explore structure-dependent hysteretic magnetizations. Then the Hopt. shift in ME response was analyzed in particulate ME composites consisting of each magnetostrictive powder  in a 0.948Na0.5K0.5NbO3–0.052LiSbO3 (NKNLS) piezoelectric matrix.In this study, the structural effects of magnetostrictive materials on ME response was investigated in order to decrease 3O4  and Fe2O3  powders were mixed by ball milling for 24 h. The well-mixed and fully dried powders were calcined at 1000 °C for 2 h. The calcined powders were ball-milled for 24 h and then sintered at 1200 °C for 2 h. After crushing and sieving of the sintered powders, CFO powders were selected with a particle size of 24–64 μm. As shown in 3O4  and Fe2O3  powders were mixed by ball milling for 24 h. Then, the well-mixed and fully dried powders were calcined at 1000 °C for 2 h without and with pelletizing at 30 bar pressure, respectively. The calcined CFO powders exhibiting a porous structure (pCFO) and a dense structure (dCFO) were mixed with 0.1 and 0.2 molar ratio of ZnO powders , respectively. Then the mixed powders were sintered at 1200 °C for 2 h. After crushing and sieving of the sintered powders, pCZFO and dCZFO powders were selected with particle sizes of 24–64 μm.2CO3 , Na2CO3 , Li2CO3 , Nb2O5 , and Sb2O5  powders were mixed by ball milling for 24 h. Then, the well-mixed and fully dried powders were calcined at 880 °C for 2 h. After sintering at 1050 °C for 2 h of CFO-NKNLS, pCZFO-NKNLS, and dCZFO-NKNLS pellets with a magnetostrictive/piezoelectric weight ratio of 0.1, disk-type ME composites were prepared with a thickness of 1 mm and a diameter of 13 mm. The ME composites were poled in silicone oil at room temperature by applying a dc field of 3 kV/mm for 30 min.ME composites were prepared with a 3-0 type particulate structure consisting of the magnetostrictive powders  in a lead-free piezoelectric matrix of NKNLS. For preparation of NKNLS powders, Kα (λ = 1.5406 Å) radiation. The surface morphology was investigated by scanning electron microscopy . Hysteretic magnetization curves were characterized by vibrating sample magnetometry . Piezoelectric constants were measured by an APC YE 2730A d33 meter . ME voltages were measured by applying an Hac of 1 Oe at an off-resonance frequency, f, of 1 kHz using a lock-in amplifier  [Hac of 1 Oe with an off-resonance frequency of 1 kHz. Then, an Hdc of ±1000 Oe was applied to the ME samples using an electromagnet to obtain reliable ME voltages. Output ac voltage (Vac) from the ME samples was measured by the lock-in amplifier.Crystal structures were investigated by X-ray diffraction  with CuKCA, USA) ,20. As s2O4 (JCPDS card No. 22-1086) [α radiation, and h, k, and l are the Miller indices. As the (311) peak shifts to a lower angle by Zn substitution, the lattice constant increases due to a decrease in the value of sin θ. With respect to the ionic radius, the pCZFO and dCZFO powders were found to exhibit an increased lattice constant compared to CFO powders because Zn2+ (0.82 Å) has a larger ionic radius than Co2+ (0.78 Å), which is replaced by Zn2+ [Crystal structures of the magnetostrictive CFO, pCZFO , and dCZFO  powders were investigated from XRD patterns. As shown in 22-1086) ,22. Even by Zn2+ ,24,25.Ms), remanent magnetization (Mr), coercive field (Hc), and magnetic susceptibility (χ = dM/dH) were investigated, as shown in Ms with decreased Hc, as shown in Ms demonstrate that the addition of Zn2+ ions causes a migration of Fe3+ ions from a tetrahedral site to an octahedral site, which causes an increase of the total magnetic moment by reducing the net magnetic moment in the tetrahedral site. Furthermore, decreased values of Hc illustrate that grain growth by Zn substitution causes an increase of the domain wall number, resulting in large grain size, which requires less energy for spin rotation [χmax of 0.22–0.42 emu/g·Oe at lower values of Hdc below ±50 Oe, compared to χmax of 0.05 emu/g·Oe at an Hdc below ±200 Oe from the CFO powders. In particular, the χmax values of dCZFO were obtained at very low values of Hdc below ±10 Oe, which are induced by prominent stepped demagnetization behavior.In terms of Zn substitution in the porous-CFO and dense-CFO powders, magnetic properties of saturation magnetization  and spinel (AB2O4) crystal structures were confirmed as piezoelectric and magnetostrictive phases, respectively. Even though sintering was conducted at 1050 °C for 2 h, all ME composites were found to exhibit stable crystal structures without any trace of secondary phase. In particular, a peak split at 2θ = 45–46° representing a tetragonal phase was maintained during the high temperature sintering. Therefore, the ME composites were found to exhibit a piezoelectric charge constant (d33) of 55–60 pC/N after sample poling.To investigate structure-dependent ME responses, particulate ME composites were prepared with compositions of CFO-NKNLS, pCZFO-NKNLS , and dCZFO-NKNLS . From the XRD patterns, as shown in αME) and Hopt. were investigated while applying Hac = 1 Oe at f = 1 kHz by sweeping Hdc of ±1000 Oe, as shown in αME = 140 μV/cm·Oe at Hopt. = 966 Oe. Even though a decreased Hopt. value of 689–828 Oe was obtained from pCZFO-NKNLS as shown in Hopt. shift due to its weak behavior of stepped demagnetization. On the other hand, the dCZFO-NKNLS composites were found to exhibit remarkable Hopt. values of 458–481 Oe as shown in Hopt. values than any reported particulate ME composites so far. As a result, the structural effect of magnetostrictive powders on Hopt. shift is clearly shown between the heterogeneous pCZFO and dCZFO powders. Although the obtained Hopt. value of 458 Oe from dCZFO-NKNLS is higher than 100 Oe, this study can serve to minimize a required Hopt. by complexation with previous studies for high feasibility of particulate ME composites.From the particulate composites of CFO-NKNLS, pCZFO-NKNLS , and dCZFO-NKNLS , ME voltage , the pCZFO and dCZFO powders were found to exhibit higher χmax of 0.22–0.42 emu/g·Oe at lower Hdc values below ±50 Oe and ±10 Oe, respectively. The NKNLS-based ME composites consisting of CFO, pCZFO, dCZFO, respectively were found to exhibit Hopt. = 966 Oe (NKNLS-CFO), Hopt. = 689–828 Oe (NKNLS-pCZFO), and Hopt. = 458–481 Oe (NKNLS-dCZFO). The results illustrate that a low Hopt. value of 458 Oe was obtained from the effective stepped demagnetization behavior of dCZFO (Zn = 0.2), which was induced by a structural effect in a heterogeneous magnetostrictive phase.In this study, magnetostrictive powders of CFO, pCZFO  and dCZFO  were prepared to produce low values of


Nitrogen (N) is a macronutrient important for the survival of plants. To investigate the effects of N deficiency, a time-course metabolic profiling of radish sprouts was performed. A total of 81 metabolites—including organic acids, inorganic acid, amino acids, sugars, sugar alcohols, amines, amide, sugar phosphates, policosanols, tocopherols, phytosterols, carotenoids, chlorophylls, and glucosinolates—were characterized. Principal component analysis and heat map showed distinction between samples grown under different N conditions, as well as with time. Using PathVisio, metabolic shift in biosynthetic pathways was visualized using the metabolite data obtained for 7 days. The amino acids associated with glucosinolates accumulated as an immediate response against –N condition. The synthesis of pigments and glucosinolates was decreased, but monosaccharides and γ-tocopherol were increased as antioxidants in radish sprouts grown in –N condition. These results indicate that in radish sprouts, response to N deficiency occurred quickly and dynamically. Thus, this metabolic phenotype reveals that radish responds quickly to N deficiency by increasing the content of soluble sugars and γ-tocopherol, which acts as a defense mechanism after the germination of radish seeds. Raphanus sativus L.), a member of the Brassicaceae family, is consumed as an edible root vegetable throughout the world. The entire plant of radish, including leaves and sprouts, is edible. It contains anthocyanins, carotenoids, flavonoids, glucosinolates, policosanols, tocopherols, and phytosterol  was edited and the log2 FC (1 DAI/0 DAI) value, and those for other days , under the two growth conditions were applied to the pathway using PathVisio. The variations in the levels of metabolites were expressed as red and green, according to the scale bar , and fructose-bisphosphate aldolase (at4g26530) genes were downregulated under severe N stress condition. Moreover, the gene for pyruvate kinase (at3g49060), the enzyme that catalyzes the final step of glycolysis, was downregulated under severe N condition [Arabidopsis, phosphofructokinase (at4g04040), fructose-bisphosphate aldolase , and phosphoglycerate kinase (at3g12780) are downregulated under N deprivation condition [The differences in the levels of metabolites and the pattern of change are shown in us study . These mondition . In protondition . Phosphoe at4g265 genes wep ≤ 0.05) between the two nutrient conditions at 0 DAI  via downregulation of alkenyl hydroxalkyl producing 2 (AOP2) transcript (at4g03060) under N starvation was investigated [Arabidopsis grown under –N condition, but were increased in the roots [The levels of methionine, tryptophan, and phenylalanine, which are precursors of glucosinolates, were high at early time points and were lower under –N condition. Moreover, the contents of other amino acids , associated with glucosinolate biosynthesis, were statistically different   reportedThe assimilation of N relative to glutamine synthetase/glutamate synthase (GS/GOGAT) cycle has previously been studied ,34. SáncArabidopsis roots and shoots show distinct temporal adaptation patterns toward N starvation. γ-tocopherol is one of the most potent antioxidant agents [Glucose, fructose, and galactose were accumulated to higher levels in sprouts grown under –N condition than they were under N sufficient condition. α- and β-tocopherols were probably converted from γ-tocopherol in both the samples, but the level of γ-tocopherol was high under –N condition. The soluble sugars were increased in tomato and cabbage under –N condition ,20. Endot agents . The higt agents . Ascorbat agents . The higt agents . The lowArabidopsis. Plants with the atapg9-1 mutant gene, involved in autophagy, showed early chlorosis [There was no distinguishable difference in the level of phytosterol in the two samples. β-sitosterol and stigmasterol are important for maintaining the structure and function of the cell membrane . In the hlorosis . The 26Shlorosis . Brassinhlorosis . BecauseWith regard to the pigments, there was only a difference in the content, but no change in the pattern was observed between the samples. Under –N condition, the levels of carotenoids and chlorophylls were low in samples grown under –N condition at 1 DAI and thereafter. The decrease in the content of pigments under –N condition has been well studied in many plants including kale, cabbage, rape, rice, sorghum, sweet potato, and tomato because of their relationship with photosynthesis and senescence ,49,50,514NO3 (10.30 mM), 0.950 g KNO3 (9.40 mM), 0.085 g KH2PO4 (0.63 mM), 0.185 g MgSO4·7H2O (10.75 mM), 0.220 g CaCl2·2H2O (1.50 mM), 0.415 mg KI (2.50 μM), 3.150 mg H3BO3 (0.05 mM), 8.450 mg MnSO4·H2O (0.05 mM), 4.300 mg ZnSO4·7H2O (0.015 mM), 0.125 mg Na2MoO4·2H2O (0.50 μM), 0.0125 mg CuSO4·5H2O (0.05 μM), 0.0125 mg CoCl2·6H2O (0.05 μM), 18.65 mg Na2·EDTA (0.05 mM), 13.90 mg FeSO4·7H2O (0.05 mM), 51.55 mg vitamin , 15 g sucrose, and 8 g agar. The pH of the medium was 5.8. The nitrogen deficient medium did not contain NH4NO3 and KNO3; instead 0.950 g KCl was added for providing K. For germination, seeds sown in two different nutrient conditions were incubated for 48 h at 25 °C in the dark. After germination, the sprouts were incubated under a 16-h light/8-h dark photoperiod and light intensity of approximately 4000 lx at 25 °C. The samples were harvested at six different times, immediately after incubation in the dark (0 DAI), and at 1, 2, 3, 5, and 7 DAI. The harvested samples were freeze-dried for six days, powdered, and stored at −80 °C until analysis.Radish seeds were purchased from Nongwoo Bio Co., Ltd. . The seeds were sterilized with 1% sodium hypochlorite containing 0.01% Tween 20 for 7 min, and 70% ethanol for 5 min. They were then rinsed five times with deionized water. The seeds were soaked by dipping in deionized water at 37 °C for 4 h and sown on 100 mL of modified Murashige and Skoog’s medium (with or without N) in Phytohealth . The composition (per L) of nutrient sufficient medium was as follows: 0.825 g NHv/v) methanol:water:chloroform were blended with 60 µL of ribitol , which was used as an internal standard (IS). The mixture was shaken at 1,200 rpm for 30 min at 37 °C using a Thermomixer Comfort . The mixture was centrifuged at 16,000× g for 3 min at 4 °C. The aqueous supernatant (800 µL) obtained after centrifugation was mixed with 400 µL of deionized water. After vortexing, the mixture was centrifuged at 16,000× g for 3 min at 4 °C. The supernatant (900 µL), thus obtained, was completely dried using a vacuum centrifuge dryer  and freeze-dried . Thereafter, 80 µL of methoxamine (MOX) reagent  was added to the concentrated sample and incubated at 30 °C, with shaking at 1200 rpm for 90 min. This was followed by addition of 80 µL of N-methyl-N-(trimethylsilyl) trifluoroacetamide  and incubation (with shaking at 1200 rpm) at 37 °C for 30 min. The separation of hydrophilic compounds was carried out using a CP-SIL 8 CB-MS column  on an Agilent 6890N Network GC system (Agilent) coupled to a Pegasus HT TOF mass spectrometer . The derivatized sample (1 μL) was injected with a split ratio of 1:25 at 230 °C and helium gas was passed at a flow rate of 1 mL/min. The column temperature was maintained at 80 °C for 2 min; it was increased at 15 °C/min to 320 °C and held at this temperature for 10 min. The ion source and transfer line temperature were set at 200 °C and 250 °C, respectively. The mass voltage was set at 1700 volt and the mass range for scanning was 85 to 600. The qualitative analysis was performed by comparison with the respective standards, possessed in an in-house library [The freeze-dried radish sprouts (10 mg) and 1 mL of 2.5:1:1 ( library . The quaw/v) ascorbic acid and 10 μg/mL 5α-cholestane as an IS. After incubation at 85 °C for 5 min, for saponification, 120 μL of 80% (w/v) KOH was added and the mixture was incubated at 85 °C for 10 min. The saponified sample was chilled on ice for 5 min, mixed with 1.5 mL hexane and deionized water, vortexed, and centrifuged at 1200× g for 5 min at 4 °C. The hexane layer was collected in a new tube and re-extracted with an equal volume of hexane. The collected hexane layer was concentrated using nitrogen gas. For derivatization, 30 μL of MSTFA and pyridine were added and incubated at 60 °C, with shaking at 1,200 rpm for 30 min. Thereafter, 1 μL of each derivatized sample was injected into an Rtx-5MS column  attached to a GC-qMS instrument . The injection temperature and split ratio were 290 °C and 10:1, respectively. The oven temperature was initially kept at 150 °C for 2 min; it was then increased at a rate of 15 °C/min to 320 °C, and maintained at this temperature for 10 min. The carrier gas, helium, was passed at a flow rate of 1 mL/min. The ion source and interface temperature were set at 230 and 280 °C, respectively. The qualitative and quantitative analyses of lipophilic compounds were performed as described by Kim et al. (2015) [The extraction of policosanols, tocopherols, and phytosterols was performed using previously described methods . The pow. (2015) .w/v) ascorbic acid). The mixture was vortexed and incubated in a water bath at 85 °C for 5 min. For saponification, 120 μL of 80% (w/v) KOH was added and the mixture was incubated at 85 °C for 10 min and cooled on ice. This was followed by addition of 1.5 mL of hexane and deionized water. As an IS, β-apo-8′-carotenal  was used. The mixture was centrifuged at 1200× g for 5 min at 4 °C and the hexane layer was transferred into a new tube. After repeating the process of adding and collecting the hexane layer, the collected hexane fractions were dried under nitrogen gas. Thereafter, 250 μL of 50:50 (v/v) dichloromethane:methanol was used as the solvent. For analysis, an Agilent 1100 series HPLC instrument (Agilent), equipped with YMC Carotenoid S-3 μm column  and photodiode array detector, was used. The chromatographic signal was measured at 450 and 286 nm. A gradient elution was performed using 92:8 (v/v) methanol:water with 10 mM ammonium acetate (solvent A) and methyl tert-butyl ether (solvent B) at a flow rate of 1 mL/min. The gradient used for elution was as follows: 0 min, 90% A/10% B; 20 min, 83% A/17% B; 29 min, 75% A/25% B; 35 min, 30% A/70% B; 40 min, 30% A/70% B; 42 min, 25% A/75% B; 45 min, 90% A/10% B; 55 min, 90% A/10% B. The column temperature was set at 40 °C. The qualitative and quantitative analyses of carotenoids were conducted using calibration curves made for the standard compounds.The method for extraction and analysis was as described by Park et al. (2014) . For theTotal chlorophylls were extracted from 10–30 mg of samples using 100% methanol at 70 °C for 30 min with Thermomixer Comfort (Eppendorf AG) at 500 rpm speed. The sample was centrifuged at 4 °C and 3000 rpm for 10 min, and the absorbance of the supernatant was measured at 666 and 653 nm to calculate the chlorophyll content using the formula mentioned by Wellburn (1994) .v/v) methanol (1.5 mL) and incubated at 69 °C. The mixture was centrifuged at, 13,000× g for 10 min at 4 °C. The supernatant was collected, and the extraction step was repeated two more times. The collected supernatant (4.5 mL) was loaded onto a prepared disposable chromatography column , which was filled with DEAE Sephadex A-25  using 0.5 M sodium acetate. As an external standard (ES), 200 μL of 2.5 mM sinigrin was loaded onto another column at the same time. After washing with 3 mL of deionized water, 70 µL of purified sulfatase (Sigma) was added to the column and incubated at 25 °C for 16 h. The desulfoglucosinolates were eluted with 2.4 mL of deionized water. The eluate, passed through the column, was filtered with a PTFE (0.20 µm) hydrophilic syringe filter  for analysis. The sample (20 µL) was injected and separated on a C18 column  using a Waters HPLC . The chromatograms were generated at 227 nm using a Waters 2998 photodiode array detector. The third distilled water and acetonitrile were used as gradient elution solvents A and B, respectively. The gradient program used was as follows: 0 min, 99% A/1% B; 18 min, 80% A/20% B; 30 min, 80% A/20% B; 35 min, 70% A/30% B; 37 min, 99% A/1% B; 47 min, 99% A/1% B. The solvent flow was 1 mL/min and column temperature was set at 40 °C. Peak identification was done using the procedure described by Baek et al. (2016) [To 100 mg of sample, we added boiling 70% (. (2016) . The con. (2016) .t-test were obtained using the SAS 9.4 software . MetaboAnalyst 4.0  was used for preparing the heat map. PathVisio 3.3.0 was downloaded from the PathVisio website (https://www.pathvisio.org) and used for visualization of the metabolite changes in metabolites.All the experiments, except for phenotypic feature measurements (n = 6), were carried out in biological triplicates. The normalization (unit variance (UV) scaling) of metabolite data and PCA were performed using SIMCA . The results of Student’s In this study, we performed metabolite profiling of radish sprouts under N sufficient and –N conditions. For the first time, we used PathVisio to present the metabolic changes in pathway diagrams in response to –N conditions in radish sprouts. To the best of our knowledge, our results show for the first time that comprehensive metabolic profiling of radish sprouts reveals rapid response after germination to –N conditions. We found that not only was there a change in the contents of metabolites with growth of sprouts, but also there was a difference in metabolite flow between N sufficient and –N conditions during the development, from germination to the formation of true leaves. Monosaccharides, pigments, and α-, β-tocopherols increased with plant growth. Low levels of amino acids and organic acids were detected in radish sprouts grown under –N conditions. On the other hand, accumulation of sugars and γ-tocopherol was detected in radish sprouts grown under –N conditions. The dramatic change in the contents of amino acids occurred within 1 DAI, even at 0 DAI (dark incubation period for germination). This means that radish seeds could recognize the surrounding nutrient situation and reacted quickly to adapt to or overcome it. An important issue for future work is to investigate the effects of N starvation on the levels of metabolites and expression of genes in the radish sprouts. Thus, future work will focus on obtaining integrated omics data from radish sprouts grown under –N conditions for short (within 1 DAI) periods. Our results provide information about the changes in the levels of metabolites during plant developmental processes and about the system of usage of N. In addition, it offers information that would help in understanding the recognition and response mechanism against N deficiency in plants.


Botryococcus braunii is an oleaginous green algae with the distinctive property of accumulating high quantities of hydrocarbons per dry weight in its colonies. Large variation in colony structure exists, yet its implications and influence of oil distribution and diffusion dynamics are not known and could not be answered due to lack of suitable in vivo methods. This publication seeks to further the understanding on oil dynamics, by investigating naturally relevant large (700–1500μm) and extra-large (1500–2500μm) sized colonies of Botryococcus braunii  in vivo, using a comprehensive approach of chemical shift selective imaging, chemical shift imaging and spin echo diffusion measurements at high magnetic field (17.6T). Hydrocarbon distribution in large colonies was found to be localised in two concentric oil layers with different thickness and concentration. Extra-large colonies were highly unstructured and oil was spread throughout colonies, but with large local variations. Interestingly, fluid channels were observed in extra-large colonies. Diffusion-weighted MRI revealed a strong correlation between colony heterogeneity, oil distribution, and diffusion dynamics in different parts of Botryococcus colonies. Differences between large and extra-large colonies were characterised by using T2 weighted MRI along with relaxation measurements. Our result, therefore, provides first non-invasive MRI means to obtain spatial information on oil distribution and diffusion dynamics in Botryococcus braunii colonies. Algae, known for their large biodiversity and range of secondary metabolites, could provide a promising solution for this challenge.Research into biofuel sources is receiving increasing attention as the general public and policymakers become aware of the need to shift from a fossil energy based economy to a more sustainable bio-based economy. The search for new fuels is driven in part by the predicted economic consequences of climate change, but also by the necessity of replacing finite resources . HoweverBotryococcus braunii,  have thhowacene . Hydrocahowacene . Algaenai race B . In addil shales –10.Botryococcus is believed to serve multiple purposes, including buoyancy control that allows for floatation . Thu. ThuB. be colony . This ise colony . Our CSSe colony ,33. ThisB. braunii colonies (1500–2500 μm diameter) show a significantly different internal makeup, namely an altogether more heterogeneous central part and less defined outer rings. These colonies correspondingly contain a more heterogeneous distribution of oil including at the central part of the colonies. The CSSI imaging of oil shows that less defined outer ring of these extra-large colonies is rich in oil content. The finding that oil is localised near the surface of both large colonies and extra-large colonies may have some implications for oil extraction from B. braunii colonies. For example, solvent-based extraction of oils for larger colonies could occur at similar efficiencies compared to smaller colonies, since the required penetration depth stays relatively equal  [T1 and T2 maps  ,35. Hype≤1500ms) . Althoug≤1500ms) . It can aps Figs .B. braunii is known to form biofilms as well as inclusion in bacterial biofilms [B. braunii, and would provide a fast biomass accumulation scenario. Importantly, it provides a plausible mode of water channel formation, arising from interstitial space between growth clusters of B. braunii. These channels are particularly well visualised when viewed as a maximum intensity projection MIP, as seen in supplementary video , MgSO4·7H2O (200 mg L-1) and K2HPO4 (104.8 mg L-1), KNO3 (1.2 g L-1), Na2O4Se (9.44 mg L-1), FeNaEDTA (20 mg L-1). Micronutrients traces consisted of CuSO4 ·5H2O (0.08 mg L-1), ZnSO4·5H2O (0.19 mg L-1), CoSO4·7H2O (0.09 mg L-1), MnSO4·H2O (1.27 mg L-1), Na2MoO4·2H2O (0.06 mg L-1), H3BO3 (2.86 mg L-1) and concentrated H2SO4 (0.01 ml L-1). KNO3 concentration was chosen so as to minimise the potential of nitrogen growth limitations. Citric acid was omitted from the medium composition, ensuring phototrophic growth due to the absence of a carbon source. All ingredients were autoclaved separately and final medium was adjusted to pH 7.2 with NaOH. Cultures were transferred to fresh medium every 2 weeks to maintain exponential growth.3 medium . The med-1 m-2. Temperature was maintained at 25±1°C under continuous sparging with ambient air. To prevent evaporation due to gas bubbling, air was wetted by sparging through distilled water prior to entering the culture vessel. Cultures were grown in high light (450 μmol s-1 m-2) under continuous illumination for 15 weeks. Under these conditions colonies achieved remarkably large and extra-large sizes (700–2500 μm). For the MRI measurements, the colonies were transferred using a 2 ml volume pipet to a glass dish, drained of excess medium and then transferred by spatula to a 5mm NMR tubes. Teflon stoppers were inserted into the tubes to prevent displacement and dehydration of the colonies. Colonies were kept in culture medium during in vivo MRI measurements.Culture illumination was provided by continuous cool LED lighting with a Correlated Colour Temperature (CCT) of 4300K and light intensity of 30 Klux or approximately 450 μmol sAll experiments were performed on a 17.6 T (750 MHz), vertical 89 mm wide bore magnet  in combination with an Avance I console. A Bruker Micro5 probe with 5mm birdcage resonator and a built-in 48 mT/m/A (1.92 T/m at 40 A) gradient system coupled to BAFPA 40A amplifiers was used for all measurements. All spectrometer operation was controlled by a Linux PC running Topspin 2.0PV and Paravision 5.1. Sample temperature was maintained at 293 ± 1 K through gradient water-cooling.2; matrix size = 256x256; and resolution = 19.5x19.5x100 μm3.Anatomical reference images were acquired using a Multiple Slice Multi Echo Sequence (MSME), refocusing magnetisation from an initial 90° pulse with a 180° pulse echo train. The following basic parameters were used for T1) and transverse (T2) relaxation maps were obtained using a Rapid Acquisition with Relaxation Enhancement with Variable TR (RAREVTR) pulse sequence. The sequence used a saturation scheme  to acquire T1 and used a multi-echo CPMG scheme  to acquire T2. The following parameters were used: TR-array = 200, 400, 800, 1500, 3000, 5500 ms; TE = 4.5, 9.0, 13.5, 18.0, 22.5, 27 ms for each TR; echo spacing = 4.5ms; RARE factor = 1; Number of averages (NA) = 16; matrix = 128x128; acquisition time = 4 hours 52 minutes. The field of view was 5x5 mm2 with a thin slice of 0.25 mm thick to prevent partial volume effects, resulting in a resolution of 39x39x250 μm3. The voxel volume was 3.8 · 10−4 mm3 and a receiver bandwidth of 100 kHz was used. A single slice was acquired to prevent interslice modulation effects. ROIs were manually defined using an Image Sequence Analysis tool package (ISA) , Transverse relaxation was calculated using the fit function: T2 = transverse relaxation time. The T1 values were determined by image sequence analysis using a fit function: 0 is the equilibrium magnetization. Built-in functions were used to generate T1 and T2 relaxation maps from the parameter fitting on a pixel by pixel basis.Longitudinal  was used to acquire selective water and fat/oil images separated on the basis of biochemical composition, as opposed to differences in  methods . A narro3 and TE = 6.2 ms. Number of averages of 3 and TE = 8.3 ms.For k-space acquisition scheme was utilised, as implemented by the Bruker ‘weighted’ measuring method, for an improved Spatial Response Function (SRF). Basic parameters were as follows: TR = 1100 ms; TE = 12 ms; Matrix = 32x32; FOV = 5x5 mm2; slice thickness = 250 μm; resolution = 156x156x250 μm3; number of scans = 45,000. Data was reconstructed into a 64x64 matrix with linear smoothing for display. Excitation and refocusing was achieved using Sinc3 pulses with a bandwidth of 8000 Hz. Echoes were captured into 2048 points in 204.80 ms, spectral resolution = 2.4 Hz per point; and Spectral width = 10 kHz (13.3 ppm). Magnetic field homogeneity in the selected volume was optimized by shimming the water resonance. A VAPOR suppression scheme of 625 ms was applied for efficient water signal saturation. Interpulse radiofrequency delay was 150, 80, 160, 80, 100, 37.2, 15 ms between seven hermite shaped CSSI modules. RF bandwidth = 900 Hz, excitation offset = -75 Hz (-0.1 ppm).Chemical Shift Imaging (CSI) was utilised in spin echo slab selective mode. CSI employs two orthogonal phase encoding steps with pulsed gradients to record a pure spectroscopic echo upon acquisition, instead of a conventional readout gradient used in imaging. A Hanning function weighted 2 effective B-values, including imaging gradients. Diffusion gradient duration (δ) of 2.5 ms was combined with 5 ms diffusion gradient separation (Δ), for a total TR and TE of 1500 and 10.1 ms respectively. To obtain sufficient SNR 16 averages were recorded resulting in a total acquisition time of 16 hours. FOV was 5x5 mm2, matrix size 96x96 and slice thickness of 0,5 mm, resulting in a resolution of 52x52x500 μm3. Receiver BW was 100 kHz.Diffusion measurements were carried out with a spin-echo pulse sequence containing a pair of mono-polar diffusion-sensitising gradients. Gradient orientations were isotopically distributed in six directions. Gradient strength ranged from 14, 406, 1182, 1723, 3123 s/mm‘lighten’ transfer mode.All experimental data were acquired and processed using Paravision 5.1  running on CentOS 3 and figures were prepared in Adobe Photoshop CC 2015.3 and Adobe Illustrator CC 2015.2 . A false colour image  was genewww.slicer.org, [3D-MSME data were exported to DICOM using Paravision 5.1, consequently reconstructed in Slicer 3D 4.8  and furIntegration of selected signal areas in magnitude mode were overlaid on MSME reference spectra using the Bruker CSI Visualisation Tool. A representative 1D spectrum was processed and exported using CSI-Tool (Bruker).A is an Offset and I the amplitude of diffusion with diffusion coefficient D. Several Regions of Interest (ROI) were selected and used for further calculations. Tabulated data containing ROI decay curves was exported for further analysis. Because the system contains two fraction, i.e., water (w) and hydrocarbons (h), normally bi-exponential fitting is required to accurately describe the system: 2, oil is not significantly attenuated because: Diffusion data was processed with a trapezoid windowing function to remove DC offset artefact (window maximum between 12.5% and 87.5% of acquisition window). Windowed results were consequently analysed using the Bruker Image Sequence Analysis Tool. Signal intensity and standard deviation were derived from the internal fitting function ‘dtraceb’: S1 Fig3 captured with a matrix of 256x256. Scale bar: 1000 μmImaging parameter used . Resolution 19.5x19.5x200 μm(TIF)Click here for additional data file.S2 FigImaging parameters used are: repetition time, 1500 ms; echo time, 8.32 ms; number of averages, 64 and resolution, 19.5x19.5x250 μm3. Receiver bandwidth used was 100 kHz. Excitation pulse of 600 Hz wide at water resonance. Inhomogeneity in the distribution of water in the centre is seen (arrowhead). The water signal was found to be low in two oil containing bands (arrows). Scale bar: 500 μm.(TIF)Click here for additional data file.S3 FigAn enlarged area of extra-large colony in (A) is shown in (B) depicting a possible septum (white arrow). Scale bar 500 μm (A), 100 μm (B).(TIF)Click here for additional data file.S4 FigIndividual images from the +Z diffusion weighting direction. TR, 1500 ms; TE, 10.1ms; 16 averages; diffusion gradient duration 2.5 ms and gradient separation of 5 ms; effective B-values range: 14, 406, 1182, 1723, 3123 s/mm2. Scale bar 500 μm.(TIF)Click here for additional data file.S5 FigBoundary model derived from integration of MSME and CSSI imaging results on morphology of the colonies seen in (TIF)Click here for additional data file.S1 VideoMovie version generated form (MP4)Click here for additional data file.S2 Video3 captured with a matrix of 256x196x196. Video length 10s, 360 frames.Maximum Intensity Projection Video (MIP) of 3D-MSME data of multiple colonies submerged in perfluordecalin (PFD), which highlights the difference in core structure of large and extra-large colonies. Processed in Slicer 3D. Time between repetitions 350 ms and echo-time 4.66 ms. Time of acquisition 4d12h18m, 29 averages. Resolution 23x23x23 μm(MP4)Click here for additional data file.


Fucus vesiculosus is a brown seaweed used in the treatment of obesity. This seaweed synthesizes various bioactive molecules, one of them being a sulfated polysaccharide known as fucoidan (FF). This polymer can easily be found commercially, and has antiadipogenic and lipolytic activity. Using differential precipitation with acetone, we obtained four fucoidan-rich fractions (F0.5/F0.9/F1.1/F2.0) from FF. These fractions contain different proportions of fucose:glucuronic acid:galactose:xylose:sulfate, and also showed different electrophoretic mobility and antioxidant activity. Using 3T3-L1 adipocytes, we found that all samples had lipolytic action, especially F2.0, which tripled the amount of glycerol in the cellular medium. Moreover, we observed that FF, F1.0, and F2.0 have antiadipogenic activity, as they inhibited the oil red staining by cells at 40%, 40%, and 50%, respectively. In addition, they decreased the expression of key proteins of adipogenic differentiation . However, F0.5 and F0.9 stimulated the oil red staining at 80% and increased the expression of these proteins. Therefore, these fucoidan fractions have an adipogenic effect. Overall, the data show that F2.0 has great potential to be used as an agent against obesity as it displays better antioxidant, lipolytic and antiadipogenic activities than the other fucoidan fractions that we tested. Fucus vesiculosus is a brown seaweed commonly found in coastal wetlands, in temperate or cold waters of the Atlantic and Pacific oceans. It was shown that F. vesiculosus intake helps women with abnormal menstrual cycles, and health problems associated with their periods , non-essential amino acids, fetal bovine serum, sodium pyruvate, and phosphate buffered saline [PBS] were purchased from Invitrogen Corporation . Monoclonal rabbit anti-CEBPα, anti-CEBPβ, anti-PPAR-γ and anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology . Other solvents and chemicals used in this study were of analytical grade.Commercial fucoidan extracted from 2 incubator.3T3-L1 preadipocyte cells were purchased from the cell bank of Rio de Janeiro, RJ, Brazil (CR089-BCRJ/UFRJ), and maintained with 10% fetal bovine serum (FBS)/DMEM containing 4.5 g/L glucose, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 mg/mL amphotericin B at 37 °C in 5% COg for 15 min at 4 °C. Acetone was added to the supernatant until a precipitated material appeared. Like this, we used the acetone volumes of 0.9, 1.1 and 2.0, calculated from the initial solution, and the operations repeated as above.Four grams of FF were solubilized into 0.25 M sodium chloride. To this solution was added increasing volumes of acetone to precipitate the different fucoidans. To obtain fucoidan F0.5, for example, 0.5 volumes of ice-cold acetone was added to this solution slowly and under gentle agitation, and then held at 4 °C for 12 h. The precipitate formed was collected by centrifugation at 8000× In order to determine the amount of sulfate, the fucoidan samples were hydrolyzed (4 N HCl for 6 h at 100 °C), and then the sulfate amount was given by turbidimetry at 500 nm by the gelatin/barium method as described early . Sodium To determine the best hydrolysis condition, the polysaccharides were hydrolyzed with 0.5, 1, 2, and 4 M, respectively, for various lengths of time , at 100 °C and the amount of reducing sugars of each condition was determined as described early . Hydroly® HPLC system from VWR-Hitachi  with a refractive index detector . A LichroCART® 250-4 column  packed with Lichrospher® 100 NH2  was coupled to the system. The sample mass used was 0.2 mg and analysis time was 25 min. The following sugars were analyzed as references: arabinose, fructose, fucose, galactose, glucose, glucosamine, glucuronic acid, mannose, and xylose.After acid hydrolysis , sugar composition was determined by a LaChrom EliteAgarose gel electrophoresis of the fucoidans was performed in 0.6% agarose gel (7.5 cm × 10 cm × 0.2 cm thick) prepared in 0.05 M 1.3-diaminopropane acetate buffer pH 9.0, as previously described . Aliquot6+ to Mo5+ by samples and subsequent formation of a green phosphate-molybdate complex at acid pH  to obtain the protein extract. The protein content was determined using the Bradford method (1976). The control corresponds to cells that were not exposed to fucoidans. For each sample, 30 μg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent transfer to polyvinylidene difluoride (PVDF) membrane . The membranes were blocked with skim milk (1%) or albumin (1%) and incubated for 18 h at 4 °C with appropriate primary antibody , in a dilution of 1:1000. After incubation with appropriate peroxidase-conjugated (anti-mouse or anti-rabbit) secondary antibody (1:1000 dilution), the detection was performed by chemiluminescence. β-actin was used as an internal control to evaluate the uniformity of protein loading and transfer. The bands were visualized with a ChemiDoc imaging system  and quantified by ImageJ software ver.1.51k .3T3-L1 cells were plated in 100 mm plates and stimulated to differentiate into adipocytes (as described above) in the presence or absence of the 200 μg/mL samples . After 15 days of differentiation the cells were suspended in the lysis buffer [50 mM Tris-HCl (pH 7.4); 1% Tween 20; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM EGTA; 1 mM Nan = 3) and from three independent assays. The pictures (p < 0.05) was applied to prove some similarities found by ANOVA. All experiments were done in triplicate and data refer to the average of three experiments done independently.All data from the experiments were expressed as mean ± standard deviation .Statistical analysis and Pearson correlation coefficient were performed using GraphPadPrismF. vesiculosus were obtained using differential precipitation with acetone. Chemical analyzes showed fucose was the major component present in all fractions whereas the relative amounts of other monosaccharides vary according to the fraction. Monosaccharide composition, agarose gel electrophoresis, and antioxidant data showed that each fraction had a different type of fucoidan. F0.5 and F0.9 have an adipogenic effect because they stimulated the lipid accumulation in 3T3-L1 adipocytes through up-regulation of C/EBPα, C/EBPβ, and PPAR gamma. On the other hand, F1.0, and F2.0 have antiadipogenic activity. In addition, all samples had lipolytic action, especially F2.0, which tripled the amount of glycerol in the cellular medium. Among these, F2.0 showed a marked effect on the attenuation of lipid accumulation, antioxidant, and lipolytic activities, and we thus recommend it as a natural antiadipogenic agent for several biotechnological applications.In this paper, four different fucoidan-rich fractions (F0.5/F0.9/F1.1/F2.0) of commercial fucoidan from