N6-methyladenosine (m6A) emerges as one of the most important modification of RNA. Bladder cancer is a common cancer type in developed countries, and hundreds of thousands of bladder cancer patients die every year.N6-methyladenosine in bladder TIC self-renewal is unknown.There are various cells in bladder tumor bulk, and a small population cells defined as tumor initiating cells (TIC) have self-renewal and differentiation capacities. Bladder TICs drive bladder tumorigenesis and metastasis, and their activities are fine regulated. However, the role of N6-methyladenosine in bladder tumors and bladder TICs. N6-methyladenosine levels are related to clinical severity and outcome. Mettl14 is lowly expressed in bladder cancer and bladder TICs. Mettl14 knockout promotes the proliferation, self-renewal, metastasis and tumor initiating capacity of bladder TICs, and Mettl14 overexpression exerts an opposite role. Mettl14 and m6A modification participate in the RNA stability of Notch1 mRNA. Notch1 m6A modification inhibits its RNA stability. Notch1 plays an essential role in bladder tumorigenesis and bladder TIC self-renewal.Here, we found a decrease of N6-methyladenosine in bladder tumorigenesis and bladder TICs, adding new layers for bladder TIC regulation and N6-methyladenosine function.This work reveals a novel role of Mettl14 and  Bladder TICs escape anoikis and initiate oncospheres in FBS-free medium, but bladder non-TICs can’t survive [Bladder cancer is a serious cancer in the world, especially in advanced countries . There a survive , 5. Like survive –8. Highl survive . DespiteN6-methyladenosine (m6A) is the most abundant modification of mRNA in human and mice, and is very conserved among species [6A modification is identified in plants, yeast, insects, virus and so on. Recently, m6A modification was also found on some non-coding RNAs, including tRNA, rRNA, lncRNA and snRNA [6A modification is reversible, which is added by methyltransferase complex and removed by m6A demethylases. m6A methyltransferase complex is comprised of METTL3, METTL4, METTL14, WTAP, VIRMA and so on [6A demethylases [6A in RNA stability is dependent on m6A readers, including YTHDF1, YTHDF2, YTHDF3 and YTHDC1 [6A modification participates in many biological processes, including spermatogenesis and embryonic development [6A modification exerts its role in tumorigenesis, proliferation and metastasis. Mettl14 is lowly expressed in hematopoietic stem cells and liver tumor cells. m6A methyltransferase Mettl14 attenuates the tumorigenesis of AML [N6-methyladenosine and related enzymes in bladder tumorigenesis and bladder TIC self-renewal are largely unknown.Like TICs in other tumors, bladder TICs are fine regulated, and the precise regulations of bladder TIC self-renewal are still largely unknown , 11. N6- species . m6A modnd snRNA . m6A modnd so on . On the thylases , 17. Thed YTHDC1 . m6A modelopment –21, circelopment , DNA damelopment , hematopelopment  and tisselopment . As for s of AML . Recentls of AML . Another6A modification and Mettl14 were lowly expressed in bladder tumorigenesis and bladder TICs. m6A level and Mettl14 expression were negatively related to the bladder cancer severity and clinical outcome. m6A modification and Mettl14 inhibited bladder tumorigenesis and bladder TIC self-renewal through Notch1 signaling, adding a new layer of bladder TIC regulation and m6A function.Here, we found that m6A antibody was purchased from Synaptic Systems. Anti-Mettl14 (ab220030) antibody and m6A quantification kit (ab185912) were from Abcam. Fluorescence-conjugated secondary antibodies were obtained from Molecular Probes Life Technologies.Anti-β-actin (cat. no. A1978) and DAPI (cat. no. 28718–90-3) were obtained from Sigma-Aldrich. Anti-mPrimary bladder cancer samples were obtained from the Departments of Urology and Henan Institute of Urology, The First Affiliated Hospital of Zhengzhou University with informed consent. The details for bladder tumors used in this work were: #1, early bladder cancer, 63 years old, female, tumor size, 23.1 × 17.3 × 16.2 mm, non-invasive, stage I, non-metastasis. #2, advanced bladder cancer, 72 years old, male, tumor size, 38.3 × 21.8 × 19.9 mm, invasive, stage IV, metastasis. #3, advanced bladder cancer, 69 years old, female, tumor size, 35.5 × 26.8 × 21.9 mm, invasive, stage III, non-metastasis. #4, early bladder cancer, 78 years old, male, tumor size, 29.2 × 18.9 × 17.2 mm, non-invasive, stage II, non-metastasis. #5, advanced bladder cancer, 75 years old, male, tumor size, 31.9 × 29.1 × 19.8 mm, non-invasive, stage II, non-metastasis #6. advanced bladder cancer, 73 years old, male, tumor size, 31.3 × 28.8 × 28.1 mm, non-invasive, stage II, non-metastasis.Primary bladder cancer cells were obtained from bladder cancer patients. For primary cell isolation, a portion of excised tumor was incubated in Hanks balanced salt solution  and transported quickly to the laboratory on ice. Then the samples were cut into small fragments, and digested in HBSS containing 0.1% type I collagenase, 0.05% type IV collagenase, 0.03% pronase, and 0.01% deoxyribonuclease at 37 °C for 30 min. The sample was filtered through 70-μm-nylon filter and centrifuged for 4 min at 50 x g in 4 °C. Bladder cancer primary cells were in precipitation, and cell survival & purification were examined. For bladder TIC enrichment, bladder cancer primary cells were incubated with CD44 antibody for FACS, and TICs were used for sphere formation assay, m6A detection and other assays.+ bladder TICs were cultured in FBS-free DMEM/F12 medium  and seeded into Corning® Costar® Ultra-Low Attachment Multiple Well Plate . Two weeks later, sphere images were taken and sphere numbers were counted. For cell line detection, 1000 T24 cells were used for sphere formation.Sphere formation assay was detected to examine the self-renewal of bladder TICs. 500 CD44Mettl14 and Notch1 knockout cells were constructed through CRISPR/Cas9 approach. For knockout, sgRNAs were designed according to online tool (http://crispr.mit.edu/) and cloned into lentiCRISPRv2, which were transfected into 293 T cells for lentivirus package, and the lentivirus was concentrated with PEG-it Virus Precipitation Solution (System Biosciences). Bladder cancer primary cells were infected with lentivirus, and the transfected cells were collected by puromycin selection. Knockout efficiency was confirmed by Western blot, and then used for sphere formation assay, transwell invasion assay or other functional assays.6A (1:500 dilution in PBS) or anti-Mettl14 (1:500 dilution in PBS) antibodies for 2 h. After washing three times, HRP-conjugated secondary (1:500 dilution in PBS) and 3,3′-diaminobenzidine were used for visualization.For immunohistochemistry, 5-μm bladder cancer sections were treated with xylene (10 min × 2), 100% alcohol (5 min × 2), 95% alcohol (5 min), 75% alcohol (5 min), PBS (5 min), 3% H2O2 (20 min) and then boiled in Tris/EDTA buffer (PH 9.0) for antigen retrieval (20 min). Then the samples were incubated with anti-m6A dot blot, RNA were extracted from bladder tumor, TICs and spheres using standard Trizol method, and then spotted onto nylon membrane. The samples were crosslink with UV treatment, and followed by m6A antibody incubation  and subsequent HRP-conjugated secondary antibody , finally the samples were detected with 3,3′-diaminobenzidine. For loading control, 0.02% methylene blue was used to stain the same RNA samples.For m+ bladder TICs and CD133− non-TICs were enriched. For detection, FlowJo software (FlowJo v10) was used for data analyses.For FACS sorting or detection, samples were incubated with Phycoerythrin (PE)-conjugated CD133 (1:300 dilution in FACS buffer) or control antibodies (1:300 dilution in FACS buffer) for 30 min on ice, and then subjected to FACS. For FACS sorting, CD1332, 1 × 103, 1 × 104 and 1 × 105 cells were injected into BALB/c nude mice for three months’ tumor formation. The ratios of bladder TICs were calculated by ELDA  with online software (http://bioinf.wehi.edu.au/software/elda/). For most experiments, two tailed unpaired Student’s t-test was used for statistical analysis.For bladder TIC ratio analysis, 10, 1 × 106A modification exerts critical roles in many biological processes. However, its role in bladder tumorigenesis and bladder TICs is unknown. In this work, we focused on the role of m6A modification in bladder tumorigenesis and bladder TICs, and we detected the content of m6A modification in bladder tumor first.As the most widely distributed RNA modification in mammalian cells, m6A modification was detected in non-tumor, early and advanced bladder tumors, and decreased m6A content was observed along with bladder tumorigenesis. Moreover, m6A modification was related to clinical severity (Fig. 6A modification in bladder cancer was also validated by RNA dot blot (Fig. 6A modification was also detected in advanced tumors by bladder cancer tissue array and immunohistochemistry (Fig. 6A modification was also related to the clinical outcome of bladder tumor patients (Fig. 6A modification content was lower in bladder tumors and related to clinical severity.mity Fig. a. The relot Fig. b, immunolot Fig. c and blalot Fig. d, e. As try Fig. e. Moreovnts Fig. f. Taken 6A modification in bladder TICs, we enriched CD44+ bladder TICs and CD44− non-TICs, and detected m6A modification. Compared with non-TICs, lower m6A modification levels were found in bladder TICs (Fig. To further examine mICs Fig. a, which ICs Fig. b, c.Fig6A modification. Compared with non-spheres, lower m6A modification levels were found in oncospheres (Fig. 6A modification in bladder cancer spheres (Fig. 6A modification was also observed in T24 spheres (Fig. 6A modification was decreased in bladder TICs.TICs can survive in sphere formation medium and sphere formation assay emerges as a standard method to enrich bladder TICs. Accordingly, sphere formation assay was performed and spheres were collected to detect mres Fig. d. RNA dores Fig. e, f. Mea6A readers in m6A modification, we then detected the expression profiles of m6A-related genes. Among these genes, Mettl14 was lowly expressed in bladder cancer, especially in advanced bladder cancer samples (Fig. Considering the importance of methyltransferases, demethylases and mles Fig. a. What’sles Fig. b. To furles Fig. c. Intereles Fig. d, e.Fig6A modification, we generated Mettl14 knockout cells through CRISPR/Cas9 approach (Fig. Mettl14 knockout led to decreased content of m6A modification, indicating the critical role of Mettl14 in m6A modification (Fig. 6A content was observed in bladder tumors (Fig. 6-adenosine methylation assay also confirmed the activity of Mettl14 in RNA m6A modification (Fig. 6A modification.To further confirm the role of Mettl14 in mach Fig. f. Mettl1ion Fig. g, h. Morors Fig. i. What’sion Fig. j. AltogeMettl14 knockout cells to perform sphere formation assay. Mettl14 knockout cells showed increased capacity of sphere formation, indicating the inhibitory role of Mettl14 in bladder TIC self-renewal (Fig. Mettl14 knockout cells contained increased bladder TICs, which confirmed the inhibitory role of Mettl14 in bladder TIC maintenance (Fig. Mettl14 knockout cells showed enhanced proliferation capacity (Fig. Mettl14 knockout cells have enhanced invasion capacity, revealing the inhibition of Mettl14 in bladder tumor invasion (Fig. Mettl14 knockout cells through in vivo tumor propagation assay. Increased tumor propagation was found in Mettl14 knockout cells (Fig. To further explore the role of Mettl14 in bladder tumorigenesis and bladder TICs, we utilized Mettl14 knockout cells were used for tumor initiation assay in vivo. Gradient Mettl14 knockout cells were injected into BALB/c nude mice for tumor initiation. Mettl14 deleted cells showed enhanced tumor formation ability (Fig. Mettl14 knockout cells (Fig. Mettl14 deleted T24 cells showed enhanced oncosphere formation capacity (Fig. Mettl14 knockout promoted the self-renewal of bladder TICs.To further explore the role of Mettl14 in tumor initiation, We then overexpressed Mettl14 in bladder TICs and examined the self-renewal. Mettl14 overexpressing cells were generated through lentivirus and confirmed by Western blot Fig. a. Mettl1Mettl14 overexpressing cells were also used for tumor-initiating assay, and impaired tumor initiation was observed Fig. h. DecreaMettl14 knockout cells, and lowly expressed in Mettl14 overexpressing cells (Fig. 6A modification in mRNA stability [Mettl14 knockout cells. Interestingly, enhanced stability of Notch1 was observed upon Mettl14 knockout (Fig. Finally we explored the functional target genes in bladder TICs. We focused on the target genes of three major pathways, including Wnt/β-catenin, Notch and Hedgehog pathways, and found Notch1 was highly expressed in lls Fig. a. The inlls Fig. b, c. Contability , 19, we Notch1 knockout cells through CRISPR/Cas9 approach (Fig. Notch1 knockout cells showed impaired sphere formation and invasion capacities, revealing the essential role of Notch1 in bladder TICs (Fig. Notch1 knockout cell, Mettl14 knockdown showed impaired role for sphere formation and metastasis, indicating the critical role of Notch1 in Mettl14 function (Fig. Although the role of Notch signaling in TICs is well-known, its role in bladder TICs remains elusive. To explore the role of Notch1 in bladder TICs and Mettl14 function, we generated ach Fig. f. ComparICs Fig. g-j. Moreion Fig. g-j. The ion Fig. k. Altoge6A modification participates in many physiological and pathological processes [6A modification in bladder tumorigenesis and bladder TICs (shown in Figs. 6A modification and Mettl14 through various functional assays, including sphere formation, transwell invasion assay, gradient tumor initiation assay, tumor propagation assay and Ki67 staining (shown in Figs. As the most abundant modification in human mRNA, mrocesses . HoweverMettl14 knockout drove the self-renewal of bladder TICs (shown in Fig. 6A-Notch1 pathway plays a critical role in bladder tumorigenesis and bladder TICs.The self-renewal of bladder TIC is precisely regulated, and the regulation mechanism is largely unknown. Here, we revealed a novel regulatory axis of bladder TICs. 6A modification and Mettl14 (Fig. Notch1 knockout cell and re-evaluated the role of Mettl14. Impaired Mettl14 role was found upon Notch1 knockout, indicating Mettl14 mainly target Notch1 to inhibit bladder TIC (Fig. Wnt/β-catenin, Notch and Hedgehog pathways were the most important signaling pathways in TICs of various tumors –32. We dknockout cells and revealed an impaired activity of bladder TICs (shown in Fig. TICs were regulated by various signaling pathways, including Wnt/β-catenin, Notch and Hedgehog pathways. Increasing works revealed the critical role of Wnt/β-catenin and Hedgehog pathway in bladder tumorigenesis and bladder TIC self-renewal. However, the role of Notch signaling in bladder tumorigenesis and TICs remains elusive. Here we generated Notch1 6A, N1-methyladenosine, m5C and pseudouridine also emerge as critical modulators in various biological processes [6A modification has been identified on mRNA and some non-coding RNA, including microRNA, lncRNA and snoRNA [6A and other modifications in non-coding RNAs also need to be further investigated.The mRNA modification is a complicated process, with various modifications for various RNA molecules. Besides mrocesses , 34. m6Ad snoRNA . Increasd snoRNA –38. The N6-methyladenosine in bladder TIC self-renewal, adding new layers of bladder TIC regulation and N6-methyladenosine function. As a novel modulator of TICs, Mettl14-N6-methyladenosine-Notch1 pathway may be a potential target for bladder TIC elimination.Bladder TICs drive bladder tumorigenesis and metastasis, and their regulation remains largely unknown. Here we revealed the role of


Aflatoxin B1 (AFB1), deoxynivalenol (DON), HT-2, ochratoxin A (OTA), zearalenone (ZEA) are the most common mycotoxins that are found in corn-based animal feed which have multiple toxic effects on animals and humans. Previous studies reported that these mycotoxins impaired mammalian oocyte quality. However, the effective concentrations of mycotoxins to animal oocytes were different.In this study we aimed to compare the sensitivity of mouse and porcine oocytes to AFB1, DON, HT-2, OTA, and ZEA for mycotoxin research. We adopted the polar body extrusion rate of mouse and porcine oocyte as the standard for the effects of mycotoxins on oocyte maturation.Our results showed that 10 μM AFB1 and 1 μM DON significantly affected porcine oocyte maturation compared with 50 μM AFB1 and 2 μM DON on mouse oocytes. However, 10 nM HT-2 significantly affected mouse oocyte maturation compared with 50 nM HT-2 on porcine oocytes. Moreover, 5 μM OTA and 10 μM ZEA significantly affected porcine oocyte maturation compared with 300 μM OTA and 50 μM ZEA on mouse oocytes. In summary, our results showed that porcine oocytes were more sensitive to AFB1, DON, OTA, and ZEA than mouse oocytes except HT-2 toxin. Mycotoxins are secondary metabolites produced by fungi, while the most agriculturally common mycotoxins known today include aflatoxins (AF), deoxynivalenol (DON), T-2, ochratoxin A (OTA), zearalenone (ZEA) . These mAflatoxins are the mycotoxins which widely exist in corn-based animal feed. Considering the toxic potency and carcinogenic action, Aflatoxin B1 (AFB1) is the most important AF . It causDeoxynivalenol that are produced mainly by fungi is a common contaminant of cereals including maize, wheat and barley . After fFusarium species. HT-2 causes a myriad of effects including the inhibition of protein, DNA and RNA synthesis; oxidative stress . TCM-199 was from Gibco . TCM-199 which contained 75 μg/ml of penicillin, 50 μg/ml of streptomycin, 0.5 μg/ml of FSH, 0.5 μg/ml of LH, 10 ng/ml of the epidermal growth factor, and 0.57 mM cysteine was used for oocyte maturation. M2 and M16 culture medium were from Sigma-Aldrich .2 for 12 h to observe the polar body extrusion.We followed the guidelines of Animal Research Institute Committee of Nanjing Agricultural University to conduct the experiments (SYXK-Su-20170007). Germinal vesicle-intact oocytes of mice that obtained from the ovaries of three- to five-week old ICR mice were collected in M2 medium and cultured with M16 medium  under the paraffin oil. These oocytes of mice were placed at 37 °C with 5% CO2 for 44 h to observe the polar body extrusion.Ovaries of Duroc pigs were purchased at a local slaughterhouse of Feng Yong Food Industry. After slaughter, ovaries were placed in a thermos bottle which contained 0.9% physiological saline and then delivered to our laboratory within 2 h. The temperature of the thermos bottle was close to 38 °C. Once the ovaries were delivered, they were washed with sterile saline. We aspirated follicular fluids from 3 to 6 mm antral follicles with a 10 ml disposable syringe and an 18 G needle. Cumulus oocyte complexes (COCs) with intact and compact cumulus were selected for maturation. These oocytes were placed at 38.5 °C with 5% COAFB1, DON, HT-2, OTA, and ZEA were dissolved and stored at 50 mM in DMSO and then diluted into different concentrations with M199 or M16 maturation medium. The GV oocytes were then cultured with these mycotoxins to analyze the maturation rate by the polar body extrusion index. The same quantity of DMSO was added in the control group.n = 3). The concentration-response curves were made by GraphPad Prism 5. At least three biological replicates were used for each analysis. Each replicate was done by an independent experiment at the different time. Results are given as means ± SEM, and two groups were compared by student t-test. A p-value of <0.05 was considered significant.Data are presented as means (n = 142) . Rates of matured oocytes were significantly decreased when the concentration of AFB1 were increased to 50 μM , 100 μM , 150 μM . We cultured the porcine oocytes for 44 h with 5, 10, 50, 100 μM AFB1. The polar body extrusion rate was 80.72 ± 7.05% (n = 217) in the control group of porcine oocytes, which was close to mouse oocytes. However, the rate of polar body extrusion in porcine oocytes was decreased with the increased AFB1 concentration. Porcine oocytes cultured with 5 μM AFB1 and the control oocytes showed no significant difference . However, the rate of polar body extrusion in porcine oocytes was significantly decreased with 10 μM , 50 μM , 100 μM  AFB1 treatment (p > 0.05), while there were significant differences when the concentrations were 10 μM (p < 0.05) and 50 μM (p < 0.01), indicating that compared with mouse oocytes, porcine oocytes were more sensitive to AFB1.We first examined the effects of AFB1 on mouse and porcine oocytes. Mouse oocytes were cultured for 12 h with 10, 50, 100, and 150 μM AFB1. The average polar body extrusion rate of the control group was 79.94 ± 4.3% (n = 142) . Comparereatment . We alson = 162) . While the rate of polar body extrusion in mouse oocytes was significantly decreased with 2 μM DON treatment , 3 μM DON treatment , 4 μM DON treatment , compared with the control group. We cultured the porcine oocytes for 44 h with 0.5, 1, 2, 3 μM DON. The polar body extrusion rate was 72.05 ± 2.6% (n = 195) in the control group of porcine oocytes, which was close to mouse oocytes. At the concentration of 0.5 μM, the control group and 0.5 μM treatment group  showed no significant difference. However, the rate of polar body extrusion in porcine oocytes was significantly decreased with 1 μM DON treatment , 2 μM DON treatment , 3 μM DON treatment  (p < 0.001), 2 μM (p < 0.01), 3 μM (p < 0.01), polar body extrusion rates of mouse oocytes and porcine oocytes showed significant difference  , when mo< 0.001) . To compfference , indicatn = 153) , 20 nM HT-2 treatment , 30 nM HT-2 treatment , 40 nM HT-2 treatment . The porcine oocytes were cultured for 44 h with 10, 50, 100, 150, 200, and 400 nM HT-2. Similar with the mouse oocytes, our results showed that HT-2 affected porcine oocyte maturation. The polar body extrusion rate was 78.19 ± 2.03% (n = 171) in the control group of porcine oocytes, which was close to mouse oocytes. However, except the 10 nM HT-2 treatment , the rate of polar body extrusion in porcine oocytes was significantly decreased with 50 nM HT-2 treatment , 100 nM HT-2 treatment , 150 nM HT-2 treatment , 200 nM HT-2 treatment , 400 nM HT-2 treatment  (p < 0.01) rates of mouse and porcine were significantly different  , while t< 0.001) . The conifferent . Our resn = 169) , while the rate of polar body extrusion in mouse oocytes was significantly decreased with 300 μM OTA treatment , 400 μM OTA treatment , 600 μM OTA treatment . The porcine oocytes were cultured for 44 h with 1, 5, 10, 30, and 100 μM OTA. Similar with the mouse oocytes, our results showed that OTA affected porcine oocyte maturation. The control group and 1 μM OTA group showed no significant difference . However, the rate of polar body extrusion in porcine oocytes was significantly decreased with 5 μM OTA treatment , 10 μM OTA treatment , 30 μM OTA treatment , 100 μM OTA treatment   , compare < 0.01) . With th < 0.01) , indicatn = 155) (n = 154) which showed no significant difference with the control groups (p > 0.05). While the rate of polar body extrusion in mouse oocytes was significantly decreased with 50 μM ZEA treatment , 100 μM ZEA treatment , 200 μM ZEA treatment . For porcine oocytes, the average polar body extrusion rate of the control groups was 77.85 ± 9.51% (n = 175), while the 5 μM ZEA groups  showed no significant differences with the control groups. However, the rate of polar body extrusion in porcine oocytes was significantly decreased with the 10 μM groups , 30 μM ZEA treatment  and 50 μM ZEA treatment  (p > 0.05). However, there were significant difference between mouse oocytes and porcine oocytes at 10 μM (p < 0.01) and 50 μM (p < 0.001)  , when th < 0.01) . Our res< 0.001) , indicatIn the present study we used the polar body extrusion as the index for oocyte maturation to compare the sensitivity of mouse and porcine oocytes to AFB1, DON, HT-2, OTA, and ZEA. Our results provided the basic database for the mycotoxin on oocyte studies.Mycotoxins are shown to affect human and animal health from multiple aspects, such as immune system, micro-organisms. And recent years, the toxicity of mycotoxins on reproductive system especially on oocytes and sperms were reported. Our previous work found that when 50 μM AFB1 affected COCs growth, especially the polar body extrusion was significantly reduced in porcine oocytes . HoweverA total of 2 μM DON was shown to affect the formation of the meiotic spindle in mouse oocytes . While aA recent study indicated that OTA significantly impaired oocyte maturation, in vitro fertilization (IVF) rates and inhibited embryonic development in vitro, because OTA could induce caspase-dependent apoptosis with in vivo model . 1–10 μMIn all, our results showed that these five mycotoxins all affected mouse and porcine oocyte quality, however, different sensitivity patterns between mouse and porcine oocytes were found. Generally porcine oocytes were more sensitive to AFB1, DON, OTA, ZEA compared with mouse oocytes except HT-2. Our results provided a basic database for the further studies on mammalian oocytes.10.7717/peerj.5111/supp-1Supplemental Information 1Click here for additional data file.


An estimated optical band gap of 1.69 eV with HOMO and LUMO levels of −5.29 eV and −3.60 eV, respectively, as well as a high thermal stability up to 398 °C were characterized for the synthesized polymer. PTPABDT fabricated as an encapsulated top gate/bottom contact (TGBC), organic field effect transistors (OFET) exhibited a p-type behavior with maximum field-effect mobility (µmax) and an on/off ratio of 1.22 × 10−3 cm2 V−1 s−1 and 7.47 × 102, respectively.In this study, we reported the synthesis and characterization of a novel hyperbranched polymer (HBPs)  Solution processable π-conjugated semiconductors, a subset of organic materials, has progressively attracted much attention from both academic and industrial applications as the best candidate for lightweight, transparent, flexible electronic devices with less production cost for high throughput processing ,2,3,4. THyperbranched polymers (HBPs) represent highly branched three-dimensional (3D) macromolecules . Their gBenzodithiophene (BDT) in particular is recognized for its symmetrical and planar structure which guarantees a more ordered π-π interactions to facilitate electron/holes transport. Moreover, BDT solubility properties and frontier energy levels are absolutely tunable since the central benzene core offers two sites for substituents attachments via alkylation reactions . On the trisdithiophene] (PTPABDT) which features a benzodithiophene (BDT) and star-shaped triphenylamine (TPA) by A3 + B2 approach, as a potential organic active layer for OFETs. As a proof of concept demonstration, the polymer was fabricated as an active layer in staggered top gate/bottom contact (TGBC) OFET. The device with PMMA as gate dielectric achieved hole mobility (µFet) of 1.22 × 10−3 cm2 V−1 s−1 and threshold voltage of −24.18 V at optimum annealing temperature of 100 °C. Taking into account of the fact that hyper branched polymer-based OFETs are uncommon, this study demonstrated that coupling triphenylamine and benzodithiophene are promising moieties to build HBPs for organic electronic application.In this research, we present a communication on the synthesis and characterization of a hyperbranched conjugated polymer Triphenylamine, toluene (anhydrous 99.9%), dimethyl formamide (anhydrous 99.9%), tetrakis(triphenylphosphine)palladium (99.9%), methanol, acetone, hexane, and chloroform were received from Sigma Aldrich . All chemicals were used as received unless stated otherwise. The monomer 2,6-bis(trimethyltin)-4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)benzodithiophene (BDT) was prep1H NMR spectroscopy. The optical spectra by UV-absorption spectroscopy was run using T60 Visible spectrophotometer  for both solution and film state. The number and weight average molecular weights of the polymers were determined by Gel Permeation Chromatography  equipped with a TDA 302 detector and a PL-gel (Varian) column, using chloroform as the eluent and polystyrene as the standard. Thermogravimetric analysis (TGA) was performed under a nitrogen atmosphere at a heating rate of 10 °C/min with a Dupont 9900 analyzer . Electrochemical properties were performed by cyclic voltammetry using in a three-electrode system with Ag/Ag+ electrode, Pt wire and 1.6 mm diameter Pt electrode as the pseudo reference, counter, and working electrodes, respectively. X-ray diffraction analysis  was performed to identify the polymer phase transformations. Nuclear magnetic resonance (NMR) spectra were recorded using Bruker DPX-300 NMR spectrometer  at room temperature with TMS as internal standard. Chemical shifts of NMR were obtained in ppm (part per million) relative to the residual solvent peak for 2 was added dropwise in the dark. Once the addition was completed, the solution was stirred for 2 h for the reaction to be completed. Organic layer was washed with water and dried over the anhydrous sodium sulfate. The solvent was removed under reduced pressure. Crude product was recrystallized in CHCl3 -hot ethanol to obtain 1.67 g product, with 84% yield. 1H NMR : δ (ppm) = 7.33 , 6.91 ; 13C NMR : δ (ppm) = 146.02, 132.50, 125.60, 116.05. Elemental analysis calculated (%) for C18H12Br3 N: C 44.85, H 2.51, N 2.91; found: C 44.81, H 2.47, N 2.93.Triphenylamine  was dissolved in 30 mL of chloroform after that the solution was cooled down to 0 °C and kept under constant stirring. Then 3 mL, 50 mmol of bromine, and Br3)4  were added to a 50 mL round-bottom flask. Then, anhydrous DMF (2 mL) and anhydrous toluene (8 mL) were added. The polymerization was carried out at 110 °C under argon protection. After 48 h, the reaction mixture was cooled to about 50 °C and added slowly to a vigorously stirred methanol (50 mL). The polymer fibers were collected by filtration. Then the crude polymer was redissolved in chlorobenzene and precipitated again into methanol. The precipitate was then subjected to Soxhlet extraction with methanol, acetone, hexane, and chloroform. The final polymers were obtained by the evaporation of chloroform and precipitating in methanol. The polymer was filtered and dried in vacuum at 40 °C for 12 h. Red material is obtained in the yield of 76%. Mn = 11.8 KDa, Mw = 22.7 KDa, PDI = 1.92. 1H NMR : δ (ppm) = 7.52 , 7.30 , 7.24 , 6.88 , 6.50 , 2.75 , 1.54 , 1.28–1.1 , 0.80 , Anal. calcd: C, 73.10; H, 6.84; N, 0.70; S, 19.35%; found C, 73.08; H, 6.80; N, 0.69; S, 19.31%.Tris(4-bromophenyl)amine , bis(trimethyltin)-4,8-bis(5-(2-ethylhexyl)thiophe n-2-yl)benzodithiophene(BDT) , and Pd was spin-coated on the PTPABDT layer followed by baking at 80 °C for 2 h in the same N2-purged globe box to remove all residual solvents. OFET devices were completed by depositing 50 nm Al top gate electrode via thermal evaporation using a metal shadow mask. Electrical characteristics of the PTPABDT devices were measured using a Keithley 4200 parameter analyzer on a probe station.After conventional photolithography process of developing photoresist layer for source/drain contact electrode pattern on a glass substrate, a 3 nm thick Ni, and 13 nm thick Au metals were deposited thorough thermal evaporation to yield channel width to lengths ratio of 1:10. Sequentially, the glass substrates were cleaned in an ultrasonic bath with deionized water, acetone, and isopropanol for 10 min each. The glass substrates were treated with UV for 30 min. 5 mg/mL solution of PTPABDT polymer was spin-coated at 2000 rpm for 60 s. Then, they were thermally annealed at 100 °C, 150 °C, and 200 °C for 30 min each on a hot plate in a N1H-NMR spectroscopy. In addition, we examined the thermal stability using the TGA analysis. The curve in Hyperbranched conjugated polymer PTPABDT which features a bifunctional benzodithiophene (BDT) and star shaped trifunctional triphenylamine (TPA) were coupled together through Stille reaction as shown in gopt) of PTPABDT was determined as 1.69 eV from the onset of the absorption edge (734 nm) of the film spectra by the relation Egopt = 1240/(wavelength of the film absorption edge).The spectra in FC is the potential of the reference redox, the Fc/Fc+ estimated as 0.25 eV vs. Ag/Ag+ in the same experimental condition. The HOMO level was determined to be −5.29 eV with LUMO level calculated as −3.60 eV from Egopt = ELUMO − EHOMO. The summary of the polymer material’s thermal, optical, and electrochemical properties are tabulated as shown in Represented in D = −20 V) and saturation (VD = −80 V) regimes. The corresponding output curve measured at VG = −20 V to −80 V with −20 V increments illustrated in D) was recorded at 100 °C. The drain current level was improved by an order of magnitude by thermal annealing, which proved to be an effective means of enhancing device performance. We attribute orderly polymer crystal rearrangement and realignment to the enhance transistor performance. We employed X-ray diffraction to investigate polymer crystallinity for the as-cast and film at the optimum annealing temperature, 100 °C. Analysis showed no distinct crystalline peak in both films as shown in −4 cm2 V−1 s−1, −16.03 ± 2.05 V in pristine OFETs to (1.05 ± 0.125) × 10−3 cm2 V−1 s−1, −24.18 ± 0.50 V in OFETs annealed at 100 °C, respectively.The synthesized PTPABDT polymer was fabricated as an active layer in staggered top gate/bottom contact (TGBC) OFET to assess the charge transport properties of the polymer. −4 cm2 V−1 s−1, 1.22 × 10−3 cm2 V−1 s−1, 1.02 × 10−3 cm2 V−1 s−1, and 7.28 × 10−4 cm2 V−1 s−1 at 0 °C, 100 °C, 150 °C, and 200 °C, respectively. The experimental mobility data is also comparable with the mobility of the triphenyl polymers reported in the literature dithiophene (BDT) and triphenylamine (TPA) moieties by A


Avelumab is a human anti-PD-L1 IgG1 monoclonal antibody that has shown antitumor activity in several advanced cancers. We report results from JAVELIN Solid Tumor JPN, a phase 1 trial of avelumab in Japanese patients with advanced solid tumors with expansion in patients with advanced gastric cancer/gastroesophageal junction cancer.In the dose-escalation part, eligible patients had various previously treated metastatic or advanced solid tumors. In the dose-expansion part, patients had stage IV gastric cancer/gastroesophageal junction adenocarcinoma and disease progression after prior therapy that included a platinum and fluoropyrimidine agent. Patients received avelumab every 2 weeks intravenously at 3, 10, or 20 mg/kg during dose escalation and 10 mg/kg during dose expansion.Among 17 patients who received avelumab in the dose-escalation part, no dose-limiting toxicities occurred, and the maximum tolerated dose was not reached. 40 patients were enrolled in the dose-expansion part, of whom 21 (52.5%) had received ≥ 3 prior lines of therapy for advanced disease. In these patients, the objective response rate was 10.0%  and median overall survival was 9.1 months . Three of 40 patients (7.5%) had a grade 3 treatment-related adverse event , and no grade ≥ 4 treatment-related adverse events occurred. Five patients (12.5%) had an immune-related adverse event .Avelumab showed acceptable safety in Japanese patients with advanced solid tumors and clinical activity in patients with advanced gastric cancer/gastroesophageal junction cancer and disease progression after chemotherapy.The online version of this article (10.1007/s10120-018-0903-1) contains supplementary material, which is available to authorized users. Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-related death worldwide . The incThe immune checkpoint proteins programmed cell death 1 ligand 1 (PD-L1) and programmed cell death 1 protein (PD-1) appear to play significant roles in GC/GEJC pathobiology, with PD-L1 being expressed in 30–45% of GC/GEJC tumors , 15. In P < 0.0001); objective response rates (ORRs) for nivolumab vs placebo were 11.2% vs 0%, respectively ; other reasons were death  and patient decision to receive a different treatment .In the dose-escalation and dose-expansion cohorts, the data cutoff date was August 10, 2016. In the dose-escalation part, 17 patients with various advanced solid tumors received avelumab Q2W at doses of 3 mg/kg (n = 35 (87.5%)] had GC and the remainder [n = 5 (12.5%)] had GEJC. HER2 status was positive in 11 patients (27.5%), negative in 20 patients (50.0%), and equivocal or not available in 9 patients (22.5%). All 40 patients had received prior therapy, including ≥ 3 or ≥ 4 prior lines for advanced disease in 21 patients (52.5%) and 7 patients (17.5%), respectively. Median duration of avelumab treatment was 2.7 months  and median follow-up was 19.3 months . Two patients (5.0%) were still receiving treatment at data cutoff. The most common reason for treatment discontinuation was disease progression [n = 33 (82.5%)]; other reasons were AE [n = 4 (10.0%)] and death [n = 1 (2.5%)].After analysis of safety and PK data, patients were enrolled in the dose-expansion part. Overall, 40 patients with advanced GC/GEJC, who had a median age of 63 years , were treated with avelumab 10 mg/kg Q2W , no patient had a DLT and the MTD was not reached. Of 17 patients in the full dose-escalation cohort, 16 patients (94.1%) had an AE of any grade, of whom 11 patients (64.7%) had a treatment-related AE (TRAE) of any grade: 3 patients (60.0%) at 3 mg/kg, 5 patients (83.3%) at 10 mg/kg, and 3 patients (50.0%) at 20 mg/kg , all of which were grade 1 or 2 and occurred in patients in the DLT analysis set. Other TRAEs occurring in ≥ 20% of patients were rash maculopapular [n = 4 (23.5%)] and stomatitis [n = 4 (23.5%)]. Two patients (11.8%) had an irAE: 1 patient (3 mg/kg) had grade 2 rash maculopapular, and 1 patient (20 mg/kg) had grade 3 aspartate aminotransferase increase and grade 1 alanine aminotransferase (ALT) increase. Two patients (11.8%) had serious AEs, which were not treatment-related, and no patient had an AE that led to death.Across all dose-escalation levels, IRRs identified via an expanded definition occurred in 5 patients  or second (n = 1) infusion, and did not lead to treatment discontinuation. Other TRAEs occurring at any grade in ≥ 10% of patients were pruritus [n = 6 (15.0%)], pyrexia [n = 5 (12.5%)], and rash [n = 4 (10.0%)]. Three patients (7.5%) had a grade 3 TRAE ; no grade ≥ 4 TRAEs occurred. Five patients (12.5%) had an irAE, all of which were grade 1 or 2 (Table n = 3) and maculopapular rash (n = 2) were the only irAEs that occurred in > 1 patient. Ten patients (25.0%) had a serious AE, and three patients (7.5%) had an AE that led to death ; none of these were treatment-related.All 40 patients in the dose-expansion cohort had an AE of any grade, of which 32 patients (80.0%) had a TRAE of any grade (Table Cmax) and area under the concentration–time curve from time 0–2 weeks (AUC0–336 h) after first administration increased in an approximately dose-proportional fashion between 3 and 20 mg/kg doses, and the estimated geometric mean apparent half-life (t1/2) ranged from 94 to 122 h (corresponding to 3.9–5.1 days). Mild drug accumulation following multiple infusions was observed , consistent with the estimated t1/2 from first administration and the Q2W dosing regimen. Median trough concentration levels (Ctrough) of avelumab showed a high degree of interpatient variability (coefficient of variation shown in Online Resource 1). Following administration of avelumab at 10.0 mg/kg, median Ctrough appeared to be higher in the dose-escalation part  than in the dose-expansion part .All 57 patients were evaluable for PK analysis. In the dose-escalation cohort (Online Resource 1), avelumab exposure in terms of maximum serum concentration observed postdose  had a confirmed objective response , comprising single patients with melanoma (3 mg/kg), esophageal squamous cell carcinoma (10 mg/kg), and GC (20 mg/kg). 11 additional patients had stable disease (SD) as best overall response.P = .065), respectively.In the dose-expansion cohort, the ORR was 10.0% , including CR in 1 patient (2.5%) and PR in 3 patients (7.5%) Table . Of the n = 40) was 9.1 months , and the 12-month OS rate was 31.0%  , and the 3-month PFS rate was 35.0%  , and no grade ≥ 4 TRAEs were reported.Ctrough, Cmax, and end-of-infusion levels, were lower than seen in previous studies with the same dose levels in non-Asian patients, likely due to the lower mean body weight and associated higher relative blood volume in Japanese patients [trough in the dose-expansion cohort, compared with that in the dose-escalation cohort at the same dose level, may have been due to different disease characteristics in patients with GC/GEJC vs other tumor types, or it may have been a sampling artifact due to the small number of patients (n = 6) who received the 10 mg/kg dose in the dose-escalation part.PK parameters, including patients . Howeverpatients , 32. TheBased on safety and PK findings from this study, and with consideration of PK and target occupancy data from an international phase 1 study of avelumab , the 10 As discussed earlier, 2 global phase 3 trials in patients with previously treated advanced GC/GEJC (KEYNOTE-061 and JAVELIN Gastric 300) that compared anti-PD-1/PD-L1 antibody treatment (pembrolizumab or avelumab) with standard second-line or third-line chemotherapy did not meet their primary endpoints , 30. HowBelow is the link to the electronic supplementary material.Supplementary material 1 (DOCX 222 KB)


AbstractLecideaaptrootii, in Lecidea sensu stricto from Swat Valley, Pakistan. It is most similar to L.fuscoatra in having an areolate thallus and black, lecideine apothecia with a persistent margin. However, L.aptrootii can be readily distinguished by having smaller ascospores (average length 8-10 µm). In phylogenetic analyses, using ITS and nuLSU rDNA sequences, L.aptrootii forms a sister-group relationship to L.grisella, which differs in having a rimose thallus.We describe here a new species,  PageBreakso far largely unknown due to lack of detailed surveys  at 8,611 m . This va surveys . Swat isgh peaks . The knogh peaks . In our Lecidea Ach. (Lecideaceae) that was originally described by Lecidea sensu lato  Schaer., L.atroviridis (Arnold) Th.Fr., L.bohlinii H. Magn, L.portensis Nádv. and L.tessellata Flörke  was performed using solvent C following standard methods  where altitude varies from 600 m to 2500 m. These areas have a moist temperate climate and remain under snow cover during winter while the summer season is accompanied by a significant amount of rainfall. Standard microscopy and spot tests  were use methods .PageBreakITS) and the nuclear large subunit (nuLSU) ribosomal DNA. The primer pair ITS1F  and LR6 (nuLSU region. Polymerase chain reactions (PCR) were performed in 12.5 µl volume per reaction using MyTaqTM Red Mix . PCR protocol for the ITS region consisted of initial denaturation of 5 min at 94 °C, 40 cycles of 30 sec at 94 °C, 30 sec at 48 °C, 1.5 min at 72 °C and a final extension of 5 min at 72 °C. PCR protocol for LSU consisted of initial denaturation of 75 sec at 94.5 °C, 35 cycles of 35 sec at 95 °C, 55 sec at 55 °C, 42 sec at 72 °C and a final extension of 10 min at 72 °C. PCR products were visualised on 1% agarose gel and cleaned using Exo SAP-IT  following the manufacturer’s instructions. Cycle sequencing reactions were performed using BigDye Terminator v3.1  with the same primers as used for the PCR amplification. The sequencing reactions were run on an ABI 3730 48-capillary electrophoresis DNA analyser sequencer according to established protocols (Applied Biosystems) at the Pritzker Laboratory for Molecular Systematics at the Field Museum, Chicago, IL, USA.We used apothecia to extract DNA with Fungal/Bacterial DNA Miniprep Kit  following the manufacturer’s instructions. Molecular data were generated for two loci: the internal transcribed spacer  Clauzade & Roux was used as the outgroup. Multiple sequence alignments for each individual locus were performed using programme MAFFT v7 with all parameters set to default values (Kotah and Standley 2013). The ends of alignments were trimmed to nearly an equal number of sites for all sequences. All gaps were treated as missing data. ITS and nuLSU sequences were concatenated in Bioedit v7.2.5 (ITS and another with combined ITS & nuLSU rDNA) were performed since only a limited amount of nuLSU data were available, whereas a larger number of ITS sequences, available in GenBank, allow for in-depth inference of the phylogenetic position of the new species.The t v7.2.5  using tht v7.2.5  employint v7.2.5 . For booTaxon classificationFungiLecidealesLecideaceaeM. Khan, A.N. Khalid, H. T. Lumbschsp. nov.82556237.1706°N, 72.3711°E, 18 Aug 2016, AN Khalid, GB-1 (Holotype LAH-35505).PAKISTAN. Khyber Pakhtunkhwa Province, Swat district, Gabin Jabba valley, 1600 m alt., Saxicolous, thallus irregularly areolate, apothecia epruinose, lecideine with persistent margin, asci with tholus, I+ blue, ascospores simple, ellipsoid with average size of 8–10 × 4.5–5.5 µm.PageBreakbrown, 8–16 µm; hymenium hyaline to olivaceous brown, (60)-70–98 µm tall; subhymenium light brown to dark brown, Hypothecium darkly pigmented throughout, Asci clavate with distinct tholus, the tip I+ blue, 8-spored, 50–68 × 12.30–16.70 µm; Ascospores simple, ellipsoid, (7)8–10(11) × (4)4.5–5.5(6) µm; paraphyses branched and net-like, 1.6–2.4 µm wide, not expanded at tips; vegetative propagules and conidiomata not seen.Thallus crustose, irregularly areolate, subcontiguous; prothallus usually indistinct, black when present; areoles flat, up to 1.2 mm in diameter and 300 µm thick, uniformity in colour from centre to edge; surface rough, not shiny, greenish-grey to light brown; Cortex not clearly differentiated, up to 31.5 μm in thickness; medulla I-, medullary hyphae thin walled, compactly arranged, 2.4–3.2 µm in diameter; photobiont layer up to 63 µm thick, algal cells 12.8–14.4 µm; apothecia black, round to irregular in outline, up to 1.5 mm in diameter, lecideine, epruinose with slightly raised, black, thin and persistent margin, frequently present, disc black, flat to slightly convex, proper exciple thin, dark brown to black; epihymenium light brown to dark O-methylperlatolic acid were detected with HPTLC.Thallus K-, KC+, C+ Red, P-, UV-. Gyrophoric acid, schizopeltic acid and 2’-The species is so far only known from two collections in the Swat district in Pakistan, where the species occurs on exposed siliceous rocks between 1600 and 1900 m altitude.PageBreakaptrootii” refers to the lichenologist André Aptroot who has contributed to the knowledge of lichen diversity in Pakistan  µm in PageBreakPageBreaklength . stricto . The new thallus . Recentln length  that oveL.fuscoatra have a differentiated black or grey margin, in contrast to the black cortex, whereas in L.aptrootii, the margins of areoles are concolorous with the areoles. This is a common feature of L.aptrootii and the recently described L.uniformis from North America  dataset  support the fact that the Pakistan collections are phylogenetically distinct from the morphologically similar L.fuscoatra and L.uniformis .


Schizochytrium sp. 31 is one of main DHA-producing strains. Recently, it was found that Schizochytrium can also synthesize OCFAs; however, contents and titers of DHA and OCFAs in Schizochytrium are still low, which limit its practical application.Docosahexaenoic acid  and odd-chain fatty acids  have attracted great interest, since they have been widely used in food and therapeutic industries, as well as chemical industry, such as biodiesel production and improvement. The oil-producing heterotrophic microalgae Schizochytrium, and relief of the inhibition resulted in improved both lipid content and the ratio of OCFAs in total fatty acids. Based on this finding, a novel strategy for elevating both DHA and OCFAs contents was established. First, the total lipid accumulation was increased by overexpressing a malic enzyme from Crypthecodinium cohnii to elevate NADPH supply. Second, the inhibition effect on acetyl-CoA carboxylase was relieved by overexpressing a codon-optimized ELO3 gene from Mortierella alpina, which encodes an elongase enzyme responsible for converting C16 into C18 fatty acids. After the above two-step engineering, contents of DHA and OCFAs were increased by 1.39- and 3.30-fold, reaching a level of 26.70 and 25.08% of dry cell weight, respectively, which are the highest contents reported so far for Schizochytrium. The titers of DHA and OCFAs were elevated by 1.08- and 2.57-fold, reaching a level of 3.54 and 3.32 g/L, respectively. Notably, the OCFAs titer achieved was 2.66-fold higher than the highest reported in Escherichia coli (1.25 g/L), implying potential value for industry application. To reveal the potential metabolic mechanism for the enhanced biosynthesis of both DHA and OCFAs, LC–MS metabolomic analysis was employed and the results showed that the pentose phosphate pathway and the glycolysis pathway were strengthened and intracellular propionyl-CoA concentration were also significantly increased in the engineered Schizochytrium, suggesting an increased supply of NADPH, acetyl-CoA, and propionyl-CoA for DHA and OCFAs accumulation.In this study, we found that acetyl-CoA carboxylase suffered from a feedback inhibition by C16-CoA in Schizochytrium. These will be valuable for improving commercial potential of Schizochytrium and guiding the engineering strategy in other fatty acids producing heterotrophic microalga.The discovery provides a new source of OCFAs production, and proposes a new strategy to improve contents and titers of both DHA and OCFAs in The online version of this article (10.1186/s13068-019-1484-x) contains supplementary material, which is available to authorized users. Docosahexaenoic acid DHA, C22:6) and odd-chain fatty acids  have attracted significant attentions because of various practical applications. DHA is an essential fatty acid that cannot be synthesized by human. It has been reported that DHA plays an important role in the development of nervous and visual systems in infants and young children. And it also has the positive effects on decreasing the risk of cardiovascular disease. Like DHA, OCFAs are also beneficial to human health. It is reported that OCFAs have clear association with lower multiple sclerosis, coronary heart disease, and type II diabetes risk  and odd-. FurtherSchizochytrium and Crypthecodinium [Schizochytrium sp. S31 [The industrial production of DHA has been carried out by heterotrophic microalgae fermentation, such as codinium , 15. Due sp. S31 .Crypthecodinium cohnii was doubled using a two-step chemical modulators based on adaptive laboratory evolution strategy [Agrobacterium-mediated transformation, electroporation, and particle bombardment have been established in Schizochytrium sp. TIO1101, Schizochytrium sp. PKU#Mn4, and Schizochytrium sp. 31, respectively [Schizochytrium sp. to improve DHA accumulation has been gradually carried out [Schizochytrium sp. PKU#Mn4 increased polyunsaturated fatty acids production by 1.37-fold [In recent years, various strategies have been adopted to enhance DHA production in microalga. For example, the DHA productivity of strategy , and Agrectively –20. Althried out . For exaEscherichia coli by increasing intracellular propionyl-CoA concentration and replacing enzymes involved in the initialization step of fatty acid elongation by enzymes with higher propionyl-CoA affinity [Yarrowia lipolytica, the ratio of OCFAs to total lipid was increased 1.65-fold by blocking the catabolic pathway of propionyl-CoA. Further genetic engineering by blocking β-oxidation, degradation of triacylglycerols, and pushing triacylglycerols biosynthesis, OCFAs content in dry cell weight was increased by 5.34-fold [Oryza sativa into E. coli to directly oxidize even chain Cn fatty acids into odd-chain Cn-1 fatty acids [E. coli can produce C7, C9, and C11 fatty alcohols by reversal of the beta-oxidation cycles with supplement of propionate in the medium [E. coli can be engineered to synthesize various odd-chain products in a controlled manner [Despite relatively few studies on OCFAs production were conducted so far, several strategies have been evaluated and the engineering of traditional fatty acid biosynthesis pathway is widely considered as an efficient strategy. For example, 1205 mg/L OCFAs were attained in affinity . Blockinty acids . In addie medium . By adapd manner .Schizochytrium sp. S31 has received much attention as it can achieve high biomass accumulation with high percent of oil and DHA content. Until now, the highest yield and productivity of DHA were achieved using Schizochytrium sp. S31 [Schizochytrium sp. S31 was much higher than those of other Schizochytrium strains [Schizochytrium sp. S31 could be an ideal candidate for producing both DHA and OCFAs. Such effort will not only provide a novel source for the production of OCFAs, but also improve commercial and industrial potential of Schizochytrium sp. S31.The oleaginous heterotrophic microalgae  sp. S31 . Actuall sp. S31 , 16, 26. strains . TherefoSchizochytrium, and converting C16 into C18 fatty acids could relieve the inhibition effect, resulting in elevated content of total lipid and the ratio of OCFAs in total fatty acids. Based on the finding, a novel strategy for elevation of both DHA and OCFAs content was successfully applied in Schizochytrium. First, the total lipid accumulation was increased by reinforcing NADPH supply through overexpression of a malic enzyme. Second, coding gene of an elongase enzyme responsible for converting C16 into C18 was overexpressed to relieve the inhibition effect on acetyl-CoA carboxylase activity. After the modification, contents and titers of both DHA and OCFAs were significantly enhanced. The study also provides useful information to engineer microalgae for other high-valued fatty acid production.In this study, inhibition effect on acetyl-CoA carboxylase activity was observed in Schizochytrium [Schizochytrium sp. 31 was prepared. The 50 or 100 μM of C16-CoA was added into the cell-free extraction and then acetyl-CoA carboxylase activity was determined. The results showed that acetyl-CoA carboxylase activity was decreased 38.24% or 44.98% in the presence of 50 or 100 μM of C16-CoA, respectively        Fig. . FurtherSchizochytrium sp. S31. And the titers and yields of biomass, lipid, DHA, and OCFAs are summarized in Table E. coli (1.25 g/L) [The schematic diagram of the changes of DHA, OCFAs, and C16:0 contents after two-step metabolic engineering is shown in Fig. .25 g/L) .Fig. 6ScThe LC–MS metabolomics have been established for quantifying metabolites related to central carbon metabolic pathway in microalgae previously in our laboratory , 38. TheAurantiochytrium sp. SD116, strengthening pentose phosphate pathway increased the proportion of DHA in total lipids [Heatmaps of targeted metabolite analysis in all samples were constructed by MultiExperiment Viewer software Fig. . It was l lipids –41; (ii)l lipids , 43;  was still much lower than that in Schizochytrium , which only used glucose as carbon source [E. coli, in addition to above issues, a key defect is phage contamination, which could result in cell death, during industrial application, in comparison with other eukaryotic microorganisms [Schizochytrium exhibited strong robustness and has already widely used for industrial production of DHA. The precursors of lipogenesis are abundant, since the oil content can reach roughly 50–55% in dry cell weight [E. coli [Schizochytrium could be a new candidate for industrial production of OCFAs.ntration , 12. Even source . For E. rganisms . Schizocl weight . In thisYarrowia lipolytica, it was reported that pentose phosphate pathway is responsible for generating NADPH for lipogenesis, while malic enzyme might not be involved in lipid accumulation [Schizochytrium sp. S31. Although the genome of Schizochytrium sp. S31 has not been sequenced, the fatty acid pathways have been well characterized previously [The primary source for lipogenic NADPH was varied in different species. In mulation –41. In oeviously , 49. Theeviously , 50–52, eviously . In thiseviously . These rin vivo and methods for alleviating the inhibition effect. In our study, in vitro study showed that acetyl-CoA carboxylase suffered from inhibition by C16-CoA, and in vivo study showed that decreasing the content of C16 fatty acid was able to elevate acetyl-CoA carboxylase activity and increasing C16 fatty acid content decreased acetyl-CoA carboxylase activity. Therefore, we concluded that there exists a feedback inhibition effect on acetyl-CoA carboxylase by C16-CoA or other forms of C16 in Schizochytrium sp. S31. We also proposed a novel strategy to release the inhibition of acetyl-CoA carboxylase by overexpressing ELO3 genes to convert C16 into C18, not by overexpressing thioesterase to hydrolyze fatty acid-ACP into free fatty acid.The lipogenesis begins by the action of acetyl-CoA carboxylase, and ends by an thioesterase for hydrolyzing C16- or C18-ACP into mature C16 or C18 fatty acids  C. reinhardtii [Literature also pointed that the similar feedback inhibition mechanism might also exist in other microalgae. For example, although no increase of lipid content has been reported by overexpressing acetyl-CoA carboxylase in nhardtii , overexpnhardtii , suggestE. coli and Y. lipolytica [We also found that the up-regulation of OCFAs content was accompanied by the slight decrease of biomass accumulation. It is a common phenomenon observed previously in polytica , 12, butSchizochytrium. First, total lipid accumulation was increased by reinforcing NADPH supply. Second, the inhibition effect on acetyl-CoA carboxylase activity was relieved to further elevate lipid content and, meanwhile, improve the ratios of OCFAs in total fatty acids. After metabolic engineering, contents and titers of DHA and OCFAs achieved a level of 26.70 and 25.08% of dry cell weight, respectively, representing the highest contents in Schizochytrium so far. Furthermore, the final strain produced 3.32 g OCFAs/L medium, which was the highest titer reported so far. DHA and OCFAs are important health products and industrial intermediates. The efforts can improve commercial potential of Schizochytrium, and provide valuable reference for production of high-value products in microalga.In this study, a two-step engineering strategy was established to increase the contents and titers of DHA and OCFAs in Schizochytrium sp. S31 (ATCC 20888) was obtained from the American Type Culture Collection  and cultivated in a basal liquid medium contained 5.0 g/L glucose, 1.0 g/L yeast extract , 1.0 g/L peptone, and 20.0 g/L sea salt . The seed cultures were cultivated in basal liquid medium at 25 °C and shaken at 180 rpm for 48 h, and 5% (v/v) of seed cultures were transferred into rich fermentation medium containing 40 g/L glucose, 5 g/L peptone, 10 g/L yeast extract, and 20 g/L sea salt.Schizochytrium genome. The malic enzyme gene (GFIV01087707.1) was amplified from the cDNA of C. cohnii ATCC 30556. Neomycin resistance gene, bleomycin resistance gene, codon-optimized Mortierella alpina ELO3 gene . CaMV35s promoter, CaMV poly(A) terminator and Nos terminator were amplified from the P1301-egfp plasmid. The 18s upstream, downstream, and α tubulin promoter were amplified from the Schizochytrium was conducted following the method described previously with modification [Transformation of fication . Ten mil660. For Nile Red analysis, 1.0 OD (OD660) cells were harvested, washed, and re-suspended in 1 mL of phosphate buffer saline (pH 7.2). After adding 10 μL Nile Red (10 mM), cells were incubated at 37 °C for 15 min and determined by a spectrophotometer . For total lipid content analysis, the method described previously was followed [Schizochytrium algae powder was extracted by solutions containing chloroform: methanol  with 0.01% butylated hydroxytoluene for three times. The extracts were then washed with 1.0 mL of solution containing 1.0 M KCl for once, and with double-distilled water. The solvents were removed using a vacuum concentrator system . Fatty acid profile analysis was conducted following the method described previously [Schizochytrium algae powder was re-suspended in solutions containing 2 mL of methanol with 3% sulfuric acid and 2 mL of chloroform. The solutions were heated at 98 °C for 2 h. After cooling, 1 mL of distilled water was added and vortexed for 20 s. GC–MS experiments were performed using an Agilent 5975 MSD/7890 instrument . The column was an HP-5MS . Ultra high purity helium was used as the carrier gas in a constant flow mode of 1 mL/min, and 1 μL of a given sample was injected in 1:2 split ratio mode. The temperature was kept at 80 °C for 2 min, and increased to 250 °C at a rate of 10 °C/min. When it reached 250 °C, the heating rate was decreased to 5 °C/min until 300 °C. The temperature was hold at 300 °C for 10 min before the analysis was terminated. NIST 11 mass spectral library  was used for identified fatty acid methyl esters. The NADPH content was measured using Coenzyme II NADP(H) content determination kit . Briefly, 7.5 OD of cells were harvested at 48 or 72 h, added with 1 mL of alkaline extract solution, and disrupted by cell disrupt apparatus. The solution was centrifuged at 14,000×g for 1 h. Five hundred microliters of supernatant was extracted and added with 500 μL of acid extract solution. The solution was centrifuged at 14,000×g for 10 min, and the supernatant extracted for NADPH content determination. The reaction agents were prepared according to the manufacture’s protocol. Distilled water was used as control. The absorbance wavelength was set at 570 nm. The equation for calculating NADPH content was: NADPH content (nmol/OD) = 0.0288 × (ΔA(sample−control) − 0.072).Cell density was determined by a UV-1750 spectrophotometer  at ODfollowed . Brieflyeviously . Briefly® III First-Strand Synthesis SuperMix kit . The cDNA was used as the template for RT-PCR analysis with the primers listed in Additional file orfA, orfB, and orfC genes were calculated using the 2−ΔΔCt method [Cell were harvested and re-suspended in Trizol reagent . The extraction of total RNA was conducted using a miRNeasy Mini Kit , and subsequent synthesis of cDNA was achieved using a SuperScriptt method . The pri660) of cells was collected at 48 and 72 h, and then washed using PBS buffer. The cell sediments were re-suspended in an extraction buffer (containing 100 mM KH2PO4/KOH (pH 7.5), 20% (v/v) glycerol), and disrupted by HNX-2 cell disruptor . The cell homogenates were centrifuged at 14,000×g  for 30 min at 4 °C, and the supernatants were used for enzyme and protein content analysis. The protein concentration of the supernatants was determined by the Bradford protein assay using Coomassie Brilliant Blue G-250  according to the manufacturer’s instructions. The activity of NADP+-dependent malic enzyme was measured following the methods described previously [l-malate, 4 mM MnCl2, and 0.23 mM TPN in 66.67 mM triethanolamine buffer at pH 7.4, and incubated at 25 °C for 10 min. The NADPH formed was determined at 340 nm. The enzyme activity was defined as the conversion of 1.0 μM of l-malate and NADP to pyruvate, CO2 and NADPH per minute at pH 7.4 at 25 °C [3, 5 mM MnCl2, and 5 mM ATP, 1 mg/mL bovine serum albumin, and 3 mg/L biotin in 100 mM phosphate buffer at pH 8.0. The mixture was incubated at 30 °C, and equal aliquots were extracted every 2 min and immediately added 10% (w/v) trifluoroacetic acid to stop the reaction. The acetyl-CoA in each aliquot reacted with oxaloacetate to form citrate and CoA-SH. The CoA-SH was determined by reacting with dithiobisnitrobenzoic acid to form a yellow compound, which was assessed spectrophotometrically at 412 nm. The enzyme activity was defined as the consumed of 1.0 μM of acetyl-CoA per min at pH 8.0 at 30 °C. For determination of the feedback inhibition on acetyl-CoA carboxylase, C16-CoA was added into the cell-free extract of Schizochytrium sp. S31 at a final concentration of 50 or 100 μM. The activities of NADP+-dependent malic enzyme and acetyl-CoA carboxylase were normalized by the protein concentration of the supernatants.The 5.0 OD  of cells were harvested at 48 and 72 h by centrifugation at 8000×g for 5 min at room temperature . The cells were immediately frozen at − 80 °C before use. The cells were added into a 900 μL of solution containing MeOH/H2O , quickly frozen in liquid nitrogen, and then thawed in room temperature. The samples were then frozen-thawed for three times. The supernatant was harvested by centrifugation at 15,000×g for 5 min at − 4 °C. The sediments were re-suspended in 500 μL of 80:20 MeOH/H2O (− 80 °C) and then repeated the above extraction process. The supernatant of the second extraction was mixed with the first, and the solvents were removed using a vacuum concentrator system . The sediment was resolved in 100 μL of distilled water. LC–MS analysis was performed on an Agilent 1260 series binary HPLC system  using a SYnergi Hydro-RP (C18) 150 mm × 2.0 mm I.D., 4 μm 80 Å particles column , coupled to an Agilent 6410 triple quadrupole mass analyser equipped with an electrospray ionization source. An aqueous solution containing 10 mM tributylamine and a 100% methanol (HPLC grade) solution were used as mobile phase A and B, respectively. The flow rate was set at 0.2 mL/min. The MS was operated in negative mode for multiple reaction monitoring development, method optimization, and sample analysis. Sample injected volume was 10 μL; capillary voltage was 4000 V; nebulizer gas flow rate was 10 L/min; nebulizer gas pressure was 50 psi; nitrogen nebulizer gas temperature was 300 °C. Data were obtained using Agilent Mass Hunter workstation LC/QQQ acquisition software (version B.04.01) and chromatographic peaks were subsequently integrated via Agilent Qualitative Analysis software (version B.04.00). Metabolomic data were normalized by interior control and cell number, and then subjected to the principal component analysis performed by SIMCA-P 11.5 software. Heatmaps of metabolomic data were created using the MultiExperiment Viewer software. For measuring the intracellular propionyl-CoA concentration, approximately 5.0 OD (OD660) of cells were harvested at 48 and 72 h, and the exaction and analysis methods followed the LC–MS metabolomic analysis described above. The standard propionyl-CoA purchased from Sigma  was used for determination of the standard curve of propionyl-CoA. The intracellular propionyl-CoA concentration was normalized by the value of OD660, which can be used for representing the biomass concentration in Schizochytrium [The LC–MS metabolomic analysis was carried out based on the method described previously . Brieflychytrium .t tests, and p < 0.05 was considered indicative of statistical significance.All experiments were conducted with at least three biological replicates. Statistical analyses were performed using two-tailed Student’s Additional file 1: Fig. S1. Comparison of acetyl-CoA carboxylase activity after adding C16-CoA into cell-free extract of Schizochytrium sp. S31. 0 represents adding 0 μM of C16-CoA; 50 represents adding 50 μM of C16-CoA; 100 represents adding 100 μM of C16-CoA. * indicated p < 0.05. ** indicated p < 0.01. Fig. S2. Expression of a codon-optimized ELO3 gene from Mortierella alpina in Schizochytrium. A) Schematic map of the ELO3 gene expression cassette, with the indicated primers. 1: 18s upstream; 2: CaMV 35s promoter; 3: ELO3 gene; 4: Nos terminator; 5: α tubulin promoter from Schizochytrium; 6: bleomycin resistance gene; 7: CYC1 terminator; 8: 18s downstream; B) Genomic PCR detection with primer 1 and primer 2. M: 1 kb marker from Vazyme company; 1: S-E strain; 2: wild type. C) RT-PCR detection with primer 3 and primer 4. M: 1 kb marker from Thermo Scientific company; 1: S-E strain; 2: wild type. Fig. S3. Expression of a malic enzyme gene from C. cohnii in Schizochytrium. A) Schematic map of the malic enzyme expression cassette, with the indicated primers. 1: 18s upstream; 2: CaMV 35s promoter; 3: neomycin resistance gene; 4: CaMV poly(A) terminator; 5: malic enzyme gene; 6: 18s downstream; B) Genomic PCR detection with primer 5 and primer 6. M: 1kb marker; 1: water; 2: wild type; 3: S-M strain; C) RT-PCR detection with primer 7 and primer 8. 1: water; 2: wild type; 3 S-M strain. Fig. S4. Malic enzyme activity and NADPH content in the wild type and S-M strains. A) The malic enzyme activity. Blue: wild type; Purple: S-M strain. * indicated p < 0.05; B) NADPH content. Blue: wild type; Purple: S-M strain. * indicated p < 0.05, **indicated p < 0.01. Fig. S5. Expression of a codon-optimized ELO3 gene from M. alpina in S-M strain. A) Schematic map of the ELO3 gene expression cassette, with the indicated primers. 1: CaMV 35s promoter; 2: ELO3 gene; 3: Nos terminator; 4: α tubulin promoter from Schizochytrium; 5: bleomycin resistance gene 6: CYC1 terminator; B) Genomic PCR detection with primer 1 and primer 2. M: 1 kb marker; 1: S-M-E strain; 2: S-M strain; C) RT-PCR detection with primer 3 and primer 4. M: 1 kb marker; 1: S-M strain; 2: S-M-E strain. Fig. S6. MS spectrums of methyl fatty acids detected among strains. Fig. S7. Principal component analysis of LC–MS targeted metabolomics of S-M and S-M-E strains. A) 48 h; B) 72 h. Purple: S-M strain; Green: S-M-E strain.Additional file 2: Table S1. Fatty acids profiles in the wild type, S-E, S-M and S-M-E strains. Table S2. LC–MS metabolomics data sets of S-M and S-M-E strains. Table S3. The production cost of DHA and OCFAs in the wild type and S-M-E strains. Table S4. Primers used in this study.Additional file 3: File S1. The sequence of codon-optimized ELO3 gene from Mortierella alpina.


A multivariate logistic regression was performed to identify factors that influenced the 30-day mortality. Correlation between NLR with other relevant factors  and fibrinogen) was examined using Spearman correlation analysis. The 30-day mortality was 19.3% (35/181) in the entire sample, 37.8% (28/74) in the high-NLR group, and 6.5% (7/107) in the low-NLR group (P < 0.001). In comparison to the low-NLR group, the high-NLR group had higher rate of intraventricular hemorrhage (29.7 vs. 16.8%), ICH volume (median 23.9 vs. 6.0 cm3) and ICH score (median 1.5 vs. 0), and lower GCS score (9.4 ± 4.5 vs. 12.9 ± 3.2). An analysis that divided the samples into three equal parts based on NLR also showed increasing 30-day mortality with incremental NLR . Kaplan-Meier curve showed higher 30-day mortality in subjects with high NLR than those with low NLR . High NLR (> 7.35) is associated with poor short-term survival in acute ICH patients.In a previous study in patients with intracranial hemorrhage (ICH), we found an association between high neutrophil-to-lymphocyte ratio (NLR) with poor short-term mortality. In the current study, this preliminary finding was validated using an independent patient cohort. A total of 181 ICH patients (from January 2016 to December 2017) were included. Diagnosis was confirmed using computed tomography (CT) in all cases. Patient survival (up to 30 days) was compared between subjects with high NLR (above the 7.35 cutoff;  Acute intracerebral hemorrhage (ICH) is associated with high disability and mortality. Increasing evidence suggests that inflammation contributes significantly to tissue damage caused by ICH. Specifically, activated inflammatory cells could release a variety of proinflammatory cytokines and proteases  increases with increasing severity of inflammatory response and has been associated with poor patient outcomes in cancers . All subjects were de-anonymized. Written informed consent was waived by the Ethics Review Board.Demographic information, past medical history, clinical data, and laboratory measures were collected from medical records. Hypertension was defined using the 2013 ESH/ESC Guidelines  the largest diameter of the hematoma; (B) the dimension of the hemorrhage perpendicular to the largest diameter as the second diameter; (C) the height of the hematoma, as calculated by multiplying the number of slices involved by the slice thickness. ICH volume was calculated as follows: ABC/2 .Based on our previous study , hospital admission at > 24 h after the first symptom (n = 2), infection within 2 weeks before ICH (n = 5), anticoagulant use within 3 months (n = 5), and leukemia (n = 1). The final analysis included 181 patients . The mean duration from disease onset to sample collection was 14.8 ± 6.9 h (range: 4–22). The total 30-day mortality was 19.3% (35/181). Demographic data and clinical features are shown in Table A total of 213 patients with acute ICH sought emergency care at our department during the study period; 32 patients were excluded due to treatment discontinuation within 24 h , middle , and highest . The 30-day mortality was 1.6, 15, and 41.7% in the groups with lowest, middle, and highest NLR, respectively . The two groups also differed significantly in the rate of IVH (29.7 vs. 16.8%), ICH volume (median 23.9 vs. 6 cm3), ICH score (median 2 vs. 0), GCS score (9.4 ± 4.5 vs. 12.9 ± 3.2), WBC (median 11.8 × 109/L vs. 8.3 × 109/L), neutrophil count (median 9.7 × 109/L vs. 5.1 × 109/L), lymphocyte count (0.8 × 109/L vs. 1.4 × 109/L), CRP (29 vs. 6 mg/L) ; the remaining 107 had low NLR (≤ 7.35). CRP and fibrinogen data were only available in 136 (75%) and 119 (66%) cases out of the 181 total cases, respectively. The 30-day mortality was 37.8% (28/74) in the high-NLR group vs. 6.5% (7/107) in the low-NLR group , GCS score, SBP, DBP, and WBC as independent variables. Selection of the factors was based previously reported association with clinical outcome in ICH patients  and GCS score .We conducted a logistic regression analysis that included NLR (high vs. low), age (≥ 80 years vs. below), IVH (presence vs. absence), ICH volume . Multivariate regression showed that high NLR is an independent risk for 30-day mortality.


Cyprinus carpio L.), one of the most economically important fish species in China, has a high susceptibility to viruses and bacteria. Thus far, in common carp, no data are available regarding the identification and immunologic function of the perforin.In the host immune system, perforin is a cytotoxic effector molecule that eliminate virus-infected and malignant cells. Moreover, some recent studies also imply the involvement of perforin in antibacterial immunity. Common carp . Moreover, in vivo or in vitro, after stimulation with polyI:C, LPS and Aeromonas hydrophila, each CcPRF was induced significantly, with differential expression dynamics.In the present study, the cDNA and genomic DNA sequences of three perforin isoform genes were cloned and characterized in common carp, named CcPRF isoforms.Our findings suggest that perforin might play significant roles in larval immune system and in the immune defense of common carp against viral and bacterial pathogens. Meantime, the differential expression dynamics seem to imply possible different cellular locations or functional differences across various The online version of this article (10.1186/s12917-018-1613-y) contains supplementary material, which is available to authorized users. In this process, it has been confirmed that perforin plays a crucial role [+ T lymphocytes [In mammals, natural killer (NK) cells and CD8ial role . Moreoveial role , 3. Untiial role , effectoial role  and somephocytes .Perforin is a 60–70 kDa pore-forming glycoprotein, which belongs to the membrane-attack-complex/perforin (MACPF) family . Mature Paralichtys olivaceus) [Ictalurus punctatus) [Oncorhynchus mykiss) [Carassius auratus langsdorfii) [Oplegnathus fasciatus) [Danio rerio) [Perforin was first characterized as a lytic pore-forming protein isolated from CTLs in 1985 . In humaivaceus) , 16, chanctatus) , rainbow mykiss) , ginbunasciatus) , 21 and o rerio) . Moreoveo rerio) , althougo rerio) , 20.Cyprinus carpio L.) is one of the most important aquaculture fish species in China and has a high susceptibility to viruses and bacteria. In common carp, although there were data suggesting the existence of a perforin/Gzm pathway like mammals [CcPRF1, CcPRF2 and CcPRF3 according to their amino acid identities with crucian carp perforins. We studied their evolution and analysed their constitutive expression patterns in healthy carps and during larvae ontogeny. Moreover, gene expression patterns of the three CcPRFs upon polyinosinic-polycytidylic acid (polyI:C) and Aeromonas hydrophila (A. hydrophila) stimulation were determined in vivo. Meanwhile, we isolated leukocytes from the head kidney and peripheral blood of common carp, and analysed polyI:C and lipopolysaccharides (LPS)-induced expression of CcPRFs and CD8α gene in vitro.Common carp , and the tissue samples, including those of the liver, spleen, head kidney, foregut, hindgut, gill, skin, brain and muscle, were isolated for RNA extraction.CcPRFs during embryonic and early larval stages, four pairs of parent fish were selected for artificial propagation, which was performed based on a previous study [To investigate gene expression patterns of three us study . FertiliTotal RNA was extracted from the tissue, embryo and larva samples using the RNAsimple Total RNA Kit . The concentration and quality of total RNA was measured using a spectrophotometer. The RNA template was subjected to reverse transcription into first-strand cDNA using the FastQuent RT Kit (with gDNase) .CcPRF1, CcPRF2 and CcPRF3, and a 405-bp cDNA fragment of CcPRF1, a 611-bp of CcPRF2 and a 1367-bp of CcPRF3 were obtained. PCR was performed using the following steps: denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min, with a final extension step of 72 °C for 10 min. Next, the full-length cDNA sequences of CcPRF1, CcPRF2 and CcPRF3 were obtained by the rapid amplification of the cDNA ends (RACE) method with a 5’-Full RACE and 3’-Full RACE Core Set Kit . The procedure was carried out as described in the user manual, and two rounds of PCR were performed to amplify the 5′ and 3′ flanking regions. The primers used for cDNA cloning are shown in Additional file To clone the cDNA sequences of perforin from common carp, three pairs of primers, PRF1-F/PRF1-R, PRF2-F/PRF2-R and PRF3-F/PRF3-R, were designed based on the conserved regions of published perforin cDNA sequences from other fish species. The first-strand cDNA synthesized by spleen-derived RNA served as a template to amplify the corresponding cDNA sequences of CcPRF genes, genomic DNA was purified from the spleen of common carp with the Genomic DNA Kit . Based on CcPRF1, CcPRF2 and CcPRF3 cDNA sequences, three, four and three primer pairs were designed respectively, and then their PCR products were analysed and sequenced. The primers used for DNA cloning are shown in Additional file To obtain the DNA sequences of three Escherichia coli cells DH-5α and sequenced by BGI China.All PCR products were analysed by 1% agarose electrophoresis, and the anticipated fragments were purified using the PCR purification kit . These fragments were then ligated into the pMD18-T vector , transformed into competent A. hydrophila was incubated in Luria-Bertani medium at 28 °C overnight under continuous shaking. A. hydrophila was inactivated in 0.5% formaldehyde at 4 °C overnight and was subsequently suspended in sterile PBS at a final density of 2.0 × 108 CFU/ml. Twenty-one carps whose body weight was 80 ± 5 g were injected intraperitoneally with 500 μl of inactivated A. hydrophila [The protocols for the immune challenges were carried out as described in previous studies. PolyI:C  was suspended in sterile phosphate-buffered saline (PBS) at a final density of 1.6 mg/ml, and twenty-one fish (body mass 80 ± 5 g) were injected intraperitoneally with polyI:C at 500 μl per fish –27. A. hdrophila .A. hydrophila-challenged carps, at 3, 6, 12, 24, 72 and 168 hpi, three fish were dissected, and the same tissues as those of polyI:C-stimulated group were selected for analysis. In addition, three un-stimulated fish served as control group.For the polyI:C-challenged carps, at 3 h, 6 h, 12 h, 24 h, 48 h, 72 h and 120 h post injection (hpi), three carp individuals were euthanized, and the liver, spleen, head kidney, foregut, hindgut and skin were sampled and kept in liquid nitrogen for total RNA extraction. For The method for isolating common carp PBLs was described in previous reports . HeparinAfter incubation overnight, drug treatment of polyI:C and LPS (10 μg/ml)  was performed in these two groups. At 3 h, 6 h, 12 h and 24 h post stimulation, the cells were harvested for RNA extraction. Additionally, the un-stimulated cells served as control group.CcPRF1, CcPRF2, CcPRF3, and CD8α were performed on a LightCycler® 96 Real-time PCR System  using TransStart® Tip Green qPCR SuperMix . The program was as follows: 94 °C for 30 s, followed by 40 cycles of 94 °C for 5 s, 58 °C for 15 s and 72 °C for 10 s. Finally, melt curve analysis was carried out to verify the presence of one single PCR product at the end of the assay. Each sample was analysed in triplicate, and the 40S ribosomal protein S11 gene served as a housekeeping gene to normalize the mRNA expression. The relative gene expression was determined using the 2-∆∆CT method [Gene expression analyses of T method , and thehttp://smart.embl-heidelberg.de/). Phylogenetic trees were generated with MEGA 6.0 using the neighbor-joining (NJ) method, in which the Jones-Taylor-Thornton (JTT) model was used as an amino acid substitution model. The protein tertiary structures were predicted by SWISS-MODEL (https://swissmodel.expasy.org/). GeneBank accession numbers used in phylogenetic and homology analyses are shown in Additional file The putative open reading frames (ORFs) and protein prediction were analysed with EditSeq within DNASTAR. The protein domains were predicted using the simple modular architecture research tool (SMART) (p < 0.05.The differences in relative gene expression between the stimulated group and control group (0 h) were conducted using two-way analysis of variance (ANOVA) in Graphpad Prism 5.0 and were considered significant when CcPRF1, CcPRF2 and CcPRF3 were 1869 bp, 1971 bp and 1966 bp , an ORF of 1767 bp encoding 588 amino acids and a 42-bp 3’-UTR. CcPRF2 comprised a 40-bp 5’-UTR, an ORF of 1677 bp encoding 558 amino acids and a 254-bp 3’-UTR. CcPRF3 was composed of a 66-bp 5’-UTR, an ORF of 1728 bp encoding 575 amino acids and a 172-bp 3’-UTR. Protein domain prediction showed that each CcPRF contained a signal peptide domain, a MACPF domain and a CalB domain  and the MACPF-domain containing protein  showed that the three arp Fig. .Fig. 3PhCcPRF1 shared the highest amino acid identities with crucian carp PRF1 (86.9%), grass carp PRF1 (83%) and zebrafish PRF1.9 (81%), CcPRF2 with ginbuna crucian carp PRF2 (89.8%) and zebrafish PRF1.3 (76.6%), and CcPRF3 with ginbuna crucian carp PRF3 (77.8%) and zebrafish PRF1.2 (71.0%). The amino acid identities of three CcPRFs with the human, mouse, chicken and green anole perforin proteins ranged from 29.2 to 36.8% (Table CcPRF had less than amino acid 40.0% identities (37.3–39.5%) with the other two isoforms.Homology analysis showed that CcPRF was expressed in all the nine tested tissues, including the liver, spleen, head kidney, foregut, hindgut, gill, skin, brain and muscle, with different expression levels in various tissues , skin (93.1%), brain (89.6%), foregut (88.6%), liver (66.7%), hindgut (48.3%) and head kidney (46.9%). In the spleen and gill, CcPRF1 mRNA levels was the highest. In contrast, CcPRF3 mRNA expression was obviously lower than CcPRF1 and CcPRF2, with the proportion of 23.7%, 13.6%, 5.4%, 4.9%, 3.0%, 1.8%, 0.5% and 0.6% in the head kidney, hindgut, spleen, foregut, gill, muscle, skin and brain, respectively. Moreover, in the nine tested tissues, the brain and skin showed the highest cumulative expression levels of the three CcPRFs.Overall, in healthy carps, gene expression of CcPRFs from blastula stage to 22 dph. The results showed that CcPRF1 had two expression peaks, with the first at 4 dph and the second at 22 dph. As for CcPRF2, after fertilization, its gene expression was up-regulated at embryonic stage and continued to increase up to 22 dph. While for CcPRF3, it displayed one expression peak at 22 dph. Obviously, the three CcPRFs displayed differential expression patterns in larvae ontogeny, while with a common expression peak at 22 dph.As shown in Fig. CcPRF1 gene expression was detected highest at 2 dpi in the liver, spleen, head kidney, foregut and hindgut, with 3.7-fold, 5.4-fold, 1.5-fold, 3.2-fold and 8.3-fold higher expression than the control group, respectively . CcPRF2 mRNA level reached peak values at 2 dpi in the liver, spleen and hindgut, with 2.1-fold, 2.2-fold and 5.8-fold up-regulation, respectively, as compared with the control group . In the head kidney, mRNA expression of CcPRF2 began to increase at 2 dpi and reached a peak at 5 dpi . With regard to CcPRF3, the mRNA expression of CcPRF3 never increased significantly in the analysed tissues, with the exception of the skin  and head kidney   Fig. . Additiovel Fig. , b.Fig. A. hydrophila stimulation, mRNA level of each CcPRF was significantly up-regulated in all the analysed tissues, with large fold-change higher expression than that in the control . Moreover, similar to polyI:C-stimulated group, the three CcPRFs showed different expression patterns in A. hydrophila-stimulated group.After CcPRFs and CD8α upon polyI:C and LPS in PBLs and HKLs.To further identify the immunologic function of perforin in common carp, we assayed gene expression of three CcPRF1, CcPRF2 and CcPRF3 reached peak values at 24 hpi, 12 hpi and 6 hpi, respectively, with 2.7-fold, 1.5-fold and 1.4-fold up-regulation, respectively, as compared with the control group . In response to LPS, CcPRF1 mRNA level reached the highest level at 24 hpi with 1.7-fold higher expression than the control group . Gene expression of CcPRF2 and CcPRF3 was up-regulated to the highest level at 6 hpi, with a 3.7-fold and 25.2-fold higher expression than that of the control group, respectively . In HKLs, gene expression of CcPRF1, CcPRF2 and CcPRF3 was up-regulated by polyI:C and increased to peak values at 24 hpi, with 3.8-fold, 5.5-fold and 3.0-fold higher expression than the control . Upon LPS stimulation, CcPRF1, CcPRF2 and CcPRF3 mRNA expression reached the highest level at 24 hpi as well, with 11.0-fold, 18.4-fold and 5.8-fold higher expression than the control group .In PBLs, after polyI:C stimulation, mRNA expression of p < 0.05). In response to LPS, CD8α mRNA expression was increased significantly, with 12.8-fold (12 hpi) higher expression than the control group . While in HKLs, upon polyI:C challenge, the gene expression of CD8α reached the highest level at 24 hpi, in which time point the expression level was 28.6-fold higher than the control . In response to LPS, CD8α mRNA levels reached the highest at 6 hpi, with 9.7-fold higher expression than the control .As for CD8α, in PBLs, polyI:C induced the gene expression of CD8α, with 1.5-fold (6 hpi) higher expression than the control group , than Japanese flounder. CcPRF2 genomic DNA comprised five exons and four introns, and the sizes of the exons located in its ORF were 503, 271, 599 and 304. Similarly, for zebrafish PRF1.3 gene, the size of the first exon located in its ORF was 503 bp as well. And for CcPRF3, the size of the last exon located in ORF was 1207 bp, which was the same as that of zebrafiah PRF1.2 gene. Notably, all these similarities were in line with the phylogenetic relationship of perforin in fish species   Fig. . HoweverCcPRFs, they showed a common expression peak at 22 dph  was incCcPRF isoform, where CcPRF2 showed broad expression across various tissues, and the other two were more tissue-specific. Additionally, differential expression dynamics were also detected during the larvae ontogeny of common carp Additional file 2:Table S2. Primers used for genomic DNA cloning. (DOCX 14 kb)Additional file 3:Table S3. Primers used for Real-time PCR. (DOCX 14 kb)Additional file 4:Table S4. GenBank accession numbers for perforin proteins. (DOCX 17 kb)Additional file 5:Table S5. GenBank accession numbers of Mpeg-1, C6, C7, C8 and C9 proteins. (DOCX 15 kb)Additional file 6:CcPRF1. (DOCX 13 kb)Intron and exon sequences of Additional file 7:CcPRF2. (DOCX 14 kb)Intron and exon sequences of Additional file 8:CcPRF3. (DOCX 13 kb)Intron and exon sequences of Additional file 9:Figure S1. Phylogenetic analysis of perforin, Mpeg-1, C6, C7, C8 and C9 polypeptides. This tree is generated using the neighbor-joining (NJ) method in MEGA 6.0. GenBank accession numbers used are shown in Additional file Additional file 10:ARRIVE  checklist.


We show that Trx-linked MSMEG_5998 degraded 63% AFB1 and native MSMEG_5998 degraded 31% after 4 h at 22 °C, indicating that the Trx-linked enzyme had a better AFB1-degrading ability. In a HepG2 cell culture model, Trx-linked MSMEG_5998 reduced DNA damage and p53-mediated apoptosis caused by AFB1 to a greater extent than the native enzyme. These findings suggest that Trx-linked MSMEG_5998 could potentially be developed to protect the liver from AFB1 damage, or as a candidate protein to reduce AFB1-related toxicity in animals.Aflatoxins are carcinogenic secondary metabolites of fungi that contaminate many staple crops and foods. Aflatoxin contamination is a worldwide problem, especially in developing countries, posing health hazards, e.g., causing aflatoxicosis and hepatocellular carcinoma, and even death. Biological solutions for aflatoxin detoxification are environmentally friendly and a cheaper alternative than chemical methods. The aims of the current study were to investigate: (1) the ability of MSMEG_5998, an aflatoxin-degrading F Aspergillus species, is a human liver carcinogen whose contamination of food is a significant risk factor for hepatocellular carcinoma pyrene exposure, which causes oxidative stress-related DNA damage  were used to drive protein production. The encoding sequence for the recombinant protein was engineered so that TrxA sequence was fused to the N-terminus of MSMEG_5998. To facilitate purification of the recombinant proteins, a C-terminal 6× His-tag was included. The entire protein expression unit was presented in FGD encoded by pET29a-FGD and MSMEG_5998 encoded by pDEST17-MSMEG_5998 were produced and purified as previously described ,19. To cE. coli BL21-DE3 competent cells . The transformed bacteria were grown in 40 mL of LB medium , with chloramphenicol added to a final concentration of 35 ng/mL to maintain the plasmid and incubated at 37 °C for 16–24 h with shaking. Then, the bacterial culture was inoculated into 500 mL of fresh chloramphenicol-containing LB medium, with the starting OD600 of 0.1. The culture was incubated at 37 °C for 1 h with shaking. When the OD600 of the culture reached 0.4–0.6, isopropyl β-d-1-thiogalactopyranoside  was added to a final concentration of 0.4 mM to induce protein production. The culture was then incubated for 2.5 h at 37 °C with shaking. After the induction, the bacterial cells were harvested by centrifugation at 8000× g for 20 min, washed with 20 mM Tris buffer (pH 7.5), and centrifuged again. The cell pellet was resuspended in 20 mL of 20 mM Tris buffer (pH 7.5) and phenylmethanesulfonyl fluoride  was added to a final concentration of 1 mM. Cells were lysed by sonication at 30% power and 120 cycles of 5 s-ON/15 s-OFF on ice . After sonication, 100 μL of the lysate was sampled (fraction T1). Additional 100 μL of the lysate was centrifuged at 8700× g for 20 min at 4 °C. After centrifugation, the supernatant was collected (fraction S1). Then, 100 μL of 20 mM Tris buffer (pH 7.5) was added to suspend the pellet (fraction P1). The remaining lysate from the sonication was centrifuged at 8700× g for 20 min at 4 °C and the supernatant was collected. Then, 100 μL of this supernatant was centrifuged at 16,000× g for 20 min at 4 °C. After centrifugation, the supernatant was removed (fraction S2) and the resulting pellet (fraction P2) was resuspended in 100 μL of 20 mM Tris buffer (pH 7.5). The centrifugation at 8700× g was used to separate non-lysed cells and cell debris from the cell lysate, while the centrifugation at 16,000× g was used to separate the insoluble proteins and inclusion bodies from the soluble proteins. The above five collected fractions  were analyzed by SDS-PAGE and western blotting.pSB1C3-5998 plasmid was used to transform g for 20 min at 4 °C. The supernatant was collected and stored at −20 °C.After the two sequential centrifugation steps described in 420, and 25 mM Tris (pH 7.4) (the latter four reactants are referred to as buffer A) were mixed, and incubated for 0, 2, 4, 6, and 8 h at 22 °C. The reaction was stopped by heating at 100 °C for 10 min. AFB1 levels were quantified by measuring the absorbance of ultraviolet light (365 nm) in a 96-well plate [The reactions were performed as previously described ,19. Briell plate . The perll plate . AFB1 waThe anti-AFB1 monoclonal antibody was obtained from Dr. Feng-Yih Yu from the Chung Shan Medical University (Taiwan) and diluted in phosphate-buffered saline . The antibody was used to coat the ELISA plate (0.1 mL/well) and the experiment was performed according to Yu et al. . Briefly2.Human hepatocellular carcinoma cell line, HepG2, was obtained from the American Type Culture Collection. The cells were cultured in the Eagle’s minimum essential medium supplemented with 10% fetal bovine serum , 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO4 HepG2 cells were seeded into 96-well plates containing 100 μL of the culture medium. After 24-h incubation, the medium was carefully removed and 100 μL of fresh medium, with or without AFB1, buffer A, and Trx-linked MSMEG_5998, was added, and the cells were incubated for 24 or 48 h. Then, cell viability was evaluated by using the MTT assay, following a previously published protocol [For the assay, 1 × 10protocol . The absCells were fixed in 3.7% formaldehyde at 25 °C for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, washed with Tris-Buffered Saline (TBS)-0.2% Tween 20, and blocked with 1% bovine serum albumin (BSA) in TBS-0.2% Tween 20. The cells were then incubated with anti-phospho-Ser 139-H2AX monoclonal antibody  at 4 °C overnight. The cells were next washed with TBS-0.2% Tween 20 and stained with anti-mouse secondary antibody  at room temperature for 1 h. The cells were washed with phosphate-buffered saline and the nuclei were stained with 1 μg/mL DAPI. Glass coverslips were mounted on the microscope slides using Clearmount . Specimens were stored up to 1 week at 4 °C before analysis.Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer  containing protease inhibitor cocktail and phosphatase inhibitor cocktail . Protein concentration was determined by using the Bio-Rad protein assay kit . Equal amounts of proteins from each sample were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane . The membrane was blocked for 1 h in TBS containing 5% nonfat milk and 0.2% Tween 20. To detect p-Chk1 (Ser 345), p-Chk2 (Thr 68), p53, phospho-p53 (Ser 20), p21, Bax, Bcl-2, PARP, and β-actin, monoclonal antibodies  were incubated with the membranes at 4 °C overnight. All antibodies, except for the anti-β-actin antibodies , were from Cell Signaling. To detect Trx-linked MSMEG_5998, monoclonal anti-6× His-tag  antibody was used. Membranes were subsequently washed for three times, 10 min each, in TBS-0.2% Tween 20, incubated with horseradish peroxidase-conjugated secondary antibody (Sigma) for 1 h, washed again three times as above, and visualized by using an enhanced luminol reagent for chemiluminescence .p < 0.05 was considered statistically significant.All data are presented as the mean ± SD. In all experiments, statistical comparisons of the different treatment groups were performed in SPSS Statistics 22 by using the Tukey post-hoc test in ANOVA. The value of