extfieldsofstudy: Biology Medicine

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Bacteriophage infections of microbiota can lead to leaky gut in an experimental rodent model

Increased intestinal permeability and translocation of gut microbiota from the intestinal lumen to the systemic circulation predispose patients to various diseases and may be one of the main triggers thereof. The role of microbiota in increased intestinal permeability is under intensive investigation. Here, we studied alterations in the host and increased intestinal permeability as a direct effect of treatment with a bacteriophage cocktail. After 10 days of challenge, the rats showed weight loss, messy hair, and decreased activity. Additionally, they displayed a significantly elevated lactulose:mannitol ratio and the level of circulating immune complexes. To our knowledge, this study demonstrates for the first time that increased intestinal permeability may be induced by bacteriophages that affect the microbiota. Electronic supplementary material The online version of this article (doi:10.1186/s13099-016-0109-1) contains supplementary material, which is available to authorized users.

Background
The human microbiota is comprised of bacteria, fungi, and viruses including bacteriophages, and is a very complex ecosystem that is in dynamic stability with each of its components and the host organism. Gut microbes colonize the majority of mucosal surfaces, and they play a primary role in host metabolism and are substantially involved in the development and normal action of the immune and neural systems, gastro-intestinal tract, and mucosal permeability [1,2]. The intestinal mucosa is a complex and extremely dynamic structure that readily adapts to a variety of signals that regulate intestinal permeability [3].
Intestinal barrier dysfunction or disruption, known as "leaky gut" syndrome, is characterized by the translocation of macromolecules, bacteria or their toxins to the lamina propria, which is implicated in the pathogenesis of numerous diseases [4]. Abnormally permeable mucosal barrier is associated with various pathologies including inflammatory bowel disease, Crohn's disease, neurodegenerative diseases, diabetes type 1, some types of cancers, cardiovascular disorders, rheumatoid arthritis, etc. [5][6][7]. It is currently known that the permeability of the mucosal barrier is regulated and influenced by numerous factors including the gut microbiota. Bacteria directly or indirectly modulate all components of intestinal permeability such as mucus integrity and trans-and paracellular transport [8].
Here, we evaluated the effect of primary lesions of the microbiota on intestinal permeability. To cause primary lesions of the microbiota, we used bacteriophages, which are believed to selectively interact with bacteria and to not affect eukaryotic cells [9,10]. The objective of this study was to assess the effect of microbiota treatment with bacteriophages on the intestinal permeability in vivo.
biological material was assumed following completion of consent forms.
The lactulose:mannitol ratio was measured as a marker of intestinal permeability 2 days before and 10 days after phage challenge as described by Meddings et al. [11]. Lactulose (L7877, Sigma-Aldrich) and mannitol (M8429, Sigma-Aldrich) were utilized for all arms of the study. For permeability testing of both probes simultaneously, animals were fasted for 4 h and then gavaged with 120 mg lactulose and 80 mg mannitol in 2 ml of water. Animals were placed in metabolic cages, and the urine passed over 24 h after the gavage was collected and assayed for the concentration of each probe by gas chromatography as described previously [12].
We measured serum circulating immune complexes (CIC) to evaluate possible systemic inflammatory response to the alterations caused by bacteriophage infection. Heparinized blood samples were collected at day 0 and day 10 from the tail vein of rats under sterile conditions. CIC were evaluated by sedimentation with a 4.0 % polyethylene glycol solution followed by spectrophotometry as described by Ramos et al. [13]. The CIC concentration was evaluated as the difference between the values of the probes before and after bacteriophage challenge.
All results are reported as the mean ± standard error (SE). Non-parametric paired Wilcoxon signed rank test was applied to analyze pre-and post-challenge differences. P < 0.05 was considered significant.

Results and discussion
This study demonstrated that increased intestinal permeability may be induced by bacteriophages that affect the microbiota. We have previously confirmed the functionality and selectivity of bacteriophage cocktails (unpublished data). Ten days of administration of a bacteriophage cocktail active against Staphylococcus spp., Streptococcus spp., Proteus spp., Pseudomonas spp., E. coli, K. pneumonia, and Salmonella spp. did not lead to apparent clinical changes in the gastrointestinal tract or abnormal stool in rats. At the same time, all animals showed weight loss, messy hair, and decreased activity starting from the fifth day of the bacteriophage treatment, which are considered to be related to the translocation of endotoxins across leaky mucosa.
We performed a lactulose-mannitol permeability test to determine whether bacteriophages may cause microbiota diseases resulting in alterations in the host organism in the form of increased intestinal permeability [11]. Thus, we compared alterations of urinary mannitol and lactulose excretion and changes in the lactulose:mannitol ratio 2 days before and on the 10th day after daily challenge with the bacteriophage cocktail. The excretion of mannitol was slightly but not significantly reduced after bacteriophage challenge as compared to before. At the same time, rats exhibited a significant increase (P < 0,05) in lactulose excretion after bacteriophage treatment as compared to before treatment. The animals displayed a significantly elevated lactulose:mannitol ratio (P < 0.05), which was considered to reflect increased intestinal permeability ( Fig. 1; Additional file 1: Table S1). The increase was at least 2,4-fold in all animals. All animals had a leaky gut with a benchmark lactulose/mannitol ratio >0.46 [12].
Next, we measured the serum concentrations of CIC to determine whether alterations caused by bacteriophages induced a systemic inflammatory reaction in the rats. The presence of CIC is an element of the normal immune response, and elevated levels of CIC are associated with different pathological conditions including intoxication [14]. As shown in Fig. 1, at the 10th day of treatment, the mean level of CIC was 2.5 times was higher than before treatment (P < 0.05), indicating endogenous intoxication, most likely caused by increased intestinal permeability and ongoing leaky gut. Untreated negative control animals that were kept under the same conditions as the treated animals did not show any changes over the study period (data not shown).
To our knowledge, this is the first study to indicate that experimental bacteriophage infection may be harmful for macro-organisms [15,16]. The pathological effect was revealed as increased intestinal permeability, and was a result of the phages' selective effect on the microbiota, without any direct effect on the host eukaryotic cells [17]. Over the last decade, the knowledge on abnormal intestinal permeability or leaky gut and increased bacterial translocation from the intestinal lumen has greatly progressed. This revealed the role of bacterial translocation as one of the main triggers in various poly-etiological diseases associated with chronic inflammation such as inflammatory bowel disease, Crohn's disease, Alzheimer's diseases, autism, diabetes, colonic neoplasia, heart failure, arrhythmias, rheumatoid arthritis, etc. [2,[5][6][7]. Our findings indicate that bacteriophages, which were previously not considered mammalian or human pathogens, can promote microbiota diseases and thus indirectly cause pathological conditions of mammals that are associated with leaky gut. These pathologies are believed not to be contagious or, at least, there currently is no reliable proof-of-concept of them being contagious [18,19]. However, taking into consideration their possible association with leaky gut, it can be assumed that their incidence and distribution may be caused by phages originating from the outer environment, because bacteriophages are widely spread and humans are constantly exposed to them [20]. Finally, infection of the microbiota by bacteriophages can be considered a new group of viral diseases of mammals. Additional studies should be conducted, including those using germ-free rats as controls to confirm the effect of the bacteriophage on the gut microbiota and employing metagenomic analyses of the changes in the gut microbial communities due to the bacteriophages, leading to a leaky gut.
Further studies will be necessary to unravel microbiota diseases and to evaluate their underlying mechanisms and roles in different human pathologies.

Additional file
Additional file 1: Table S1. Changes in lactulose and mannitol excretions after bacteriophage challenge.  Table S1


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Expression, purification and biological characterization of the extracellular domain of CD40 from Pichia pastoris

Background CD40, also called Bp50, is a novel member of the TNF receptor superfamily. Based on its important role in multiple physiological and pathological processes, the CD40 signaling pathway has become a vital target for treating transplantation, autoimmune diseases and cancers. This study generated a protein fragment that disrupts this signaling pathway. Results A DNA fragment encoding the extracellular domain of CD40 (CD40-N) has been codon-optimized and cloned into pPIC9K to create a Pichia pastoris expression and secretion strain. SDS-PAGE and Western blotting assays using the culture media from methanol-induced expression strains showed that recombinant CD40-N, a 27 kDa glycosylated protein, was secreted into the culture broth. The recombinant protein was purified to more than 90 % using Sephadex G-50 size-exclusion chromatography and Q Sepharose Fast Flow ion exchange. Finally, 120 mg of the protein was obtained at a relatively high purity from 3 l supernatant. Binding assay (ITC200 assay) shown the direct interaction of CD40-N and CD40 agonist antibody (G28-5). The bioactivity of recombinant CD40-N was confirmed by its ability to disrupt non-canonical NF-κB signaling activated by CD40 agonist antibody or CD40 ligand and to inhibit ant-CD40 agonist antibody-induced TNF-alpha expression in BJAB cells in vitro. In addition, our data indicate that the protein has curative potential in treating dextran sulfate sodium (DSS)-induced colitis in vivo. Conclusions The results show that the experimental procedure we have developed using P. pastoris can be used to produce large amounts of active CD40-N for research and industrial purposes. The protein fragment we have acquired has potential to be used in research or even treating inflammation diseases such as colitis.

Background
CD40 is a 50-kDa transmembrane protein that belongs to the TNF receptor family. It is not only expressed on antigen-presenting cells such as B cells, dendritic cells and macrophages, but is also found on endothelial cells, mast cells, fibroblast cells, tumor cells and smooth muscle cells, suggesting that it has extensive functions in different physiological contexts [1][2][3][4].
There are many downstream signaling pathways coupled to the CD40 intracellular region, such as Jak3-stat3, traf6-Erk, p38, JNK, and NF-κB signaling pathway [5]. Upon binding to CD40 ligand CD40 leads to NF-κB2 p100 processing into p52 and activates non-canonical NF-κB signaling, this is likely to be important for the transcriptional regulation of CD40 target genes in adaptive immune responses [6,7].
During the activation of immune responses, both TCR-MHC antigen signaling and co-stimulatory signals such as B7-CD28 are required for antigen-presenting cells to activate T cells. Activated T cells express high level of CD40 ligand that interacts with CD40 on antigen-presenting cells. In turn CD40-activated noncanonical NF-κB signaling up-regulates B7 expression on antigen-presenting cells, thus promoting antigen presentation [8][9][10][11]. In addition to the antigenpresenting process, CD40-activated signals are also involved in the priming of T cells [12], the cytotoxicity of T cells [13], the proliferation and differentiation of B cells, and immunoglobulin class switching and so on [14]. Although it participates in physiological processes, many studies have been published on its role in the pathology of disease. Activation of CD40 signaling is present in type 1 diabetes, multiple sclerosis (MS), inflammatory bowel disease (IBD), psoriasis, rheumatoid arthritis, and systemic lupus erythematosus (SLE) [15]. Disrupting the pathway has shown significant effects on the treatment of most of these diseases in mouse models (NOD, EAE, IBD, CIA and SLE mouse models) [16]. Several CD40L-CD40 interactionblocking antibodies such as BG9588, IDEC-131 and ch5D12 have gone through or are undergoing clinical trials, and some have shown curative effects. Thus, the CD40 signaling pathway is considered to be a promising target for the clinical treatment of autoimmune diseases [17][18][19].
In this study, we aimed to disrupt the CD40L-CD40 interaction by expressing the extracellular domain of CD40. CD40-N, a 174 amino acids soluble form of the extracellular domain of CD40, was designed. A methylotrophic yeast called Pichia pastoris was used in this study as an efficient protein expression system to produce large amounts (g/L) of heterologous protein [20]. The induced protein was secreted into the culture supernatant and purified by size-exclusion chromatography and ion exchange chromatography. Finally, purified CD40-N was obtained with a purity of more than 90 %. The purified protein was able to block the CD40 activated signaling in vitro and to decrease the symptom of DSS-induced colitis in vivo. Thus, the purified CD40-N protein may be useful for further functional and structural studies.

Mice
Male C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). All mice were housed and maintained in SPF conditions. All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Biomedical Research Ethics Committee of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Strains, plasmids
The cell strain GS115 and the reconstructed plasmid pPIC9K were provided by the Key Laboratory of Molecular Medicine of Fudan University [21]. The E. coli strain DH5α was purchased from TIANGEN Biotech Co., Ltd (Beijing), and pcDNA3.3 was purchased from Invitrogen.
Yeast nitrogen base (with or without ammonium sulfate) was obtained from Sigma. Other reagents were of analytical purity. Sephadex G-50, and Q-Sepharose-FF were purchased from GE Healthcare.
Construction of expression vector pPIC9K/CD40-N CD40-N is the region from 61 bp to 579 bp in CD40 (NM_001250.4), encoding amino acids 21 to 193. A codon-optimized version of CD40-N was synthesized with XhoI and NotI sites at either end and cloned into the pUC57 plasmid by Sangon Biotech. The plasmid was digested with XhoI and NotI (Thermo Scientific) to release the CD40-N sequence. The sequence was then inserted into the yeast expression vector pPIC9K using the same restriction sites. The ligation product was transformed into E. coli DH5α competent cells. Successful recombinant colonies with pPIC9k/CD40-N were confirmed by restriction digest with XhoI and NotI and sequencing. Small-scale plasmid preparations, restriction digests, ligations and transformations were performed according to the manufacturer's protocols.
Transformation of P. pastoris to produce a CD40-Nexpressing strain The constructed plasmid pPIC9K/CD40-N was linearized with SalI. The digested product was purified using an EasyPure PCR Purification Kit (Transgene Biotech) and used to transform the yeast host strain GS115. The transformation was carried out by electroporating P. pastoris as described in the Pichia expression manual (Invitrogen). Briefly, the GS115 cells were cultured in YPD medium until the OD 600 reached 0.6-0.8. Then, the cells were pelleted by centrifugation at 3000 rpm for 5 min. Competent cells were generated by washing the cells twice with ice-cold water and followed by washing twice with ice-cold D-sorbitol buffer (1 M). Finally, the competent cells were resuspended in 1 mL of D-sorbitol buffer mixed with linearized plasmid in an electroporation cuvette on ice before electroporation (Micropulser™ Bio-Rad). Transformed cells were supplied with 1 mL ice-cold D-sorbitol immediately after electroporation and cultured at 30°C for 1 h. The transformants were plated on MD plates (2 % glucose, 4 × 10 −5 % biotin, and 1.34 % YNB) for 2-3 days.
Approximately 800 colonies on the MD plate were selected and screened for G418 (Amresco E859-5G) resistance. First, colonies were synchronized twice by culturing in 200 μL YPD medium in a 96-well plate for 24 h. Then, colonies were screened in media containing 1 mg/mL G418 for 24 h. Positive colonies (those that grew on the G418 plate) were cultured in a new plate with medium containing a higher concentration of G418 (2 mg/mL) for 24 h. This procedure was repeated until the strain could not grow on the plate. Strains that could grow at the highest concentration of G418 were stored at −80°C for further experiments.
To induce the expression of CD40-N, each clone was streaked onto an YPD plate to obtain single colony. The single colony was then inoculated in 50 mL of BMGY in 250 mL flasks and cultured at 30°C with 220 rpm shaking. When the OD 600 reached 3-4, cells were harvested by centrifugation and briefly rinsed with water to remove trace glycerol. Rinsed cells were centrifuged and re-suspended in 50 mL BMMY. The cells were cultured in a new 250-mL flask at 30°C with 220 rpm shaking and supplemented with methanol to a final concentration of 1 % every 24 h. 80 μL supernatant sample was collected every 24 h to examine the expression of CD40-N by SDS-PAGE and Western blotting.

Large-scale production of CD40-N
Large-scale production of CD40-N was carried out using the clone that had the best yield in the smallscale experiments. A single colony was selected and grown in 5 mL of YPD medium at 30°C with 220 rpm shaking overnight. The overnight culture was diluted (1:40) into 200 mL YPD medium and grown at 30°C, 220 rpm shaking until an OD 600 of 4.0 was reached. The culture was transferred into 3 L of medium in a bioreactor (Bioflow 3000 NBS) and grown in batch mode for 20 h. A sharp increase in dissolved oxygen (DO) occurred when the OD 600 reached 70, suggesting that the glycerol was exhausted. Glycerol (50 %, v/v) was fed at a rate of 20 mL/(L · h) until the OD 600 reached 110. The methanol-fed phase began once all of the glycerol was consumed. The methanol feed rate gradually increased from 0.8 to 4 mL/(L · h) in the first 6 h, allowing the culture to adapt to methanol consumption. After 6 h, the methanol feed rate was maintained at 4 mL/L . h for an additional 30 h.

Purification of the CD40-N protein
The supernatant from the fermentation was loaded into an ultra-filtration system (Merck Millipore, P2B005A05) to concentrate it to approximately 500 mL. Then, the sample was run through a Sephadex G-50 size-exclusion column that had been preequilibrated with at least 2 CV (column volumes) of buffer A (20 mM Tris-HCl, pH 7.4). Then, the fraction containing the protein was applied to a Q-Sepharose-FF (2 cm × 50 cm) column at 5 mL/min using an ÄKTA explorer 100. The column was washed with buffer A until the UV 280 (nm) was at the base level. Then, a linear gradient of buffer B (1.0 mol/L NaCl, 20 mM Tris-HCl pH 7.4) was used to elute the protein from the column. The protein concentration was measured by the BCA assay. The protein sample was lyophilized and stored at −80°C.
Coomassie blue staining and western blots SDS-PAGE analysis was performed using 12 % gels according to the standard method. The entire gel was stained in Coomassie blue staining solution overnight before being placed in destaining buffer. For Western blotting, the proteins on the gel were transferred to a polyvinylidene difluoride membrane (Immobilon P, Millipore) using a wet electroblotting apparatus (Bio-Rad) at 100 V for 50 min in a solution of Trisglycine (25 mM Tris, 192 mM glycine). The membrane was blocked by incubating with 5 % non-fat milk for 1 h at RT. Then, the membrane was immunoblotted with primary antibody at 4°C overnight and with HRP-conjugated secondary antibodies for 1 h at RT. Detection of the bound antibody was performed using Super Signal West Pico Chemiluminescent Substrate (Pierce). The primary antibody against CD40-N (AF632) was purchased from R&D Systems. The anti-Flag antibody was purchased from Sigma (F3165). Mouse monoclonal antibodies against CD40 (G28-5 and 3A8) were purchased from ATCC. Anti-NF-κB2 (#4882) was purchased from Cell Signaling Technology (Danvers, MA). Anti-glyceraldehyde-3phosphate dehydrogenase (GAPDH) monoclonal antibody was purchased from Kangchen (KC-5G4, Shanghai, China).

Sugar content analysis of the recombinant protein
Purified CD40-N was treated with PNGase F (New England Biolabs P0704S) according to the manufacturer's protocol, and the treated protein was analyzed by SDS-PAGE.

ITC 200 protein protein interaction assay
The cell is filled with G28-5 at 1 mg/mL, and the syringe is filled with CD40-N at 6 mg/mL. At special time intervals (150 s), a small volume (2 μL) of the CD40-N solution is injected into the cell triggering the binding reaction and producing the characteristic peak sequence in the recorded signal (Fig. 6a), during time of each drop was 4 s, and 19 drops were injected.
Biological activity assay BJAB cells were plated at a density of 10 6 cells/mL and cultured in RPMI 1640 medium supplemented with 10 % FBS and 2-mercaptoethanol (Invitrogen) in 12-well plates. CD40-N was added at different concentrations of 0, 100, 300, 500 μg/mL at the same time with G28-5 (10 μg/mL). 1 h later, cells were harvested and qRT-PCR was performed to detect the RNA levels of Bcl-xL and TNF-alpha. CD40L was purchased from Peprotech (310-02).

RNA extraction and Real-time polymerase chain reaction
Total RNA was isolated from cell lines using Superfec-TRITM, Total RNA Isolation Reagent (Shanghai Pufei Biotech Co., Ltd, 3101-100) according to the manufacturer's protocol. To obtain cDNA, reverse transcript was performed using PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa, RR047A) according to the manufacturer's instructions and 400 ng RNA was used as template. Quantitative real-time PCR (qRT-PCR) were performed using a 7500 Fast Realtime PCR System (Applied Biosystems, Carlsbad, CA), and all qRT-PCR reagents and consumables were purchased from Applied Biosystems and TaKaRa. For each reaction, reverse transcript product was diluted 10 times and 5 μL of the products was added to a 20 μL reaction system (TaKaRa, RR420A). Other reagents including predesigned and synthesized forward and reverse primer were added according to the manufacturer's protocol and a two steps method was performed. Each sample was analyzed in triple replication. Relative quantification (RQ) was derived from the difference in cycle threshold (Ct) between target gene and actin (△Ct) using the equation RQ = 2 -△Ct . The levels of mRNA were quantitatively assessed by SYBR Green-based quantitative PCR with gene specific primers. Actin was used as control. The primers were as follow: human ACTIN forward primer CTGGAACGGTGAAGGTGACA, human ACTIN reverse primer AAGGGACTTCCTGTAACAATGCA; human Bcl-xL forward primer CTGCTGCATTGTTCCCATAG, human Bcl-xL reverse primer GACGAGTTTGAACTGCGGTA; human TNF-alpha forward primer CAGAGGGAAGAGTTCCCCAG human TNF-alpha reverse primer CCTTGGTCTGGTAGGAGACG.
Error bars represent SD, and statistical significance calculated using two-tailed, unpaired t test.

DSS-induced colitis
Male C57BL/6 mice were fed for 5 days with drinking water containing dextran sulfate sodium (DSS) (M.W. 36000-50000 Da; MP Biomedical, #160110) at a concentration of 2.75 % (w/v), and then allowed to recover by drinking regular water for another 2 days. The animals were weighted daily and monitored for signs of rectal bleeding.

Construction, expression, and detection of CD40-N
The DNA fragment encoding the partial human CD40 gene (21-193 aa), CD40-N, underwent codon optimization according to yeast's preference and was inserted between the XhoI and NotI sites of the expression vector pPIC9K. The correct sequence of the recombinant was confirmed by DNA sequencing.
Transformation of P. pastoris with pPIC9K yielded 4 transformants that were able to grow in the presence of a high concentration of geneticin, including three strains that grew in 4 mg/mL geneticin on YPD plates and one strain that grew in 1 mg/mL geneticin. After methanol induction, the supernatants were harvested and analyzed by Coomassie blue staining and Western blotting. Coomassie blue staining detected an increasing band at approximately 27 kDa that peaked at 96 h, suggesting that CD40-N may be secreted into the culture medium (the theoretical size of CD40-N is 19.3 kDa; Fig. 1a). Western blots were performed after CD40specific antibody (AF632) was checked. A human CD40-N (not undergoing codon optimization, with or without signal sequence) gene fragment was cloned into pcDNA3.3 plasmid with a Flag tag at the C-terminus. The constructs were transferred to HEK293T cells and expressed for the Western blot assay. The CD40 antibody detected the same band with the Flag antibody, demonstrating the specificity of the antibody (Fig. 1b). The antibody was used to detect the culture medium, and Western blotting results showed that the potential CD40-N band in Coomassie blue staining was specifically recognized by the CD40-specific antibody, indicating that the protein expressed by P. pastoris was recombinant CD40-N (Fig. 1c). Comparing the band intensities of different clones by Coomassie blue staining and Western blot analysis showed that clone number 1 had the highest expression level of CD40-N.
Fermentation and purification of CD40-N P. pastoris clone NO.1 was grown in a 5 l stirred bioreactor, as described in the Methods section, growth curve of the yeast was shown in Fig. 2a the amounts of expression of CD40-N was peaked at 36 h and then be kept stable (Fig. 2b). After 36 h of fermentation, the supernatant, approximately 3 l, was concentrated to approximately 500 mL after ultrafiltration using a 5 kDa membrane. Then, the 500 mL sample was applied to a flow-through Sephadex G50 size-exclusion column to be desalted and purified from smaller proteins. After that, ion-exchange chromatography was used to obtain highpurity protein. A total of 120 mg protein with a purity of more than 90 % was collected at the washing step using approximately 0.03 M NaCl in buffer Tris-HCl (pH 7.4) [22] (Fig. 3).

Sugar content of the recombinant protein
To study the glycosylation of the purified CD40-N, the purified protein was treated with the deglycosylating enzyme PNGase F, which is derived from Flavobacterium meningosepticum and can remove N-linked carbohydrates. As shown in Fig. 4, Coomassie blue staining detected a single band at a molecular weight of 27 kDa before deglycosylation and a band of approximately 19.3 kDa after treatment. This indicates that the purified CD40-N was N-glycosylated. The band at approximately 35 kDa was PNGase F [23].

ITC assay proved the interaction of CD40-N and G28-5
To study whether G28-5, a CD40 agonist antibody, can bind CD40-N directly. We use ITC 200 system to analysis the interaction [24]. The process was performed according to the method. After saturating the macromolecule, the residue heat effects are due to mechanical and dilution effects (Fig. 5a). After the integration of the area of each Fermentation supernatants were collected at the indicated times after methanol induction, and recombinant CD40-N protein is indicated with an arrow peak, the individual heats are plotted against the molar ratio from which through nonlinear regression (Fig. 5b), the thermodynamic parameters was calculated, K D =0.546 μmol, and △H =−23.24KJ/mol.

CD40-N can disrupt CD40-activated signaling
The recognition of CD40 by CD40 ligand or CD40spcific agonist antibody results in NFκB p100 processing into NFκB p52, activates alternative NFκB signaling and induces downstream target gene expression. To determine whether the purified protein CD40-N can disrupt the interaction of CD40 with CD40L or CD40-specific agonist antibody, we treated BJAB cells with CD40L in the presence of titrated CD40N. Western Blot showed that CD40-N reduced the p100 processing into p52 induced by CD40L in BJAB cells in a dose-dependent manner (Fig. 6a). G28-5 can activate CD40 signaling and further induce the expression of downstream target genes such as TNF-alpha. We observed that CD40-N significantly reduced TNF-alpha mRNA level induced by CD40-specific agonist antibody, G28-5, in a dose dependent manner (Fig. 6b), while the RNA levels of Bcl-xL was not induced by G28-5 as negative control. These data demonstrate that the purified protein CD40-N could disrupt the interaction of CD40 with CD40L or CD40-specific agonist antibody in vitro.
Functional CD40-N can relieve the symptoms of DSS-induced colitis CD40 signaling has been shown to play an important role in inflammations such as colitis [25][26][27][28]. To determine whether CD40-N functions in vivo, we employed a  DSS-induced colitis mouse model to test it. At 7-day post-treatment, the mean body weight of the PBS treated mice was reduced to 76.8 % of the starting weight; while the CD40-N treatment could significantly recover the loss of body weight (88.4 %, Fig. 7a). H&E staining indicated that damage to the architecture of the colon in CD40-N treatment mice was reduced compared to that of the control mice, and crypt atrophy and cilia damage were both less prevalent (Fig. 7b). These data reveal that CD40-N reduced inflammation in vivo to alleviate the symptoms of DSS-induced colitis in mice.

Discussion
Targeting CD40L-CD40 interaction could be useful in clinical applications for curing autoimmune diseases, providing treatment following transplantation and treating tumors [15]. One strategy to disrupt this interaction is to use an anti-CD40L monoclonal antibody: this approach has been shown to be effective in mouse models of RA, SLE, MS, IBD, T1 diabetes, and inflammatory heart disease [29]. The humanized CD40L monoclonal antibody BG9588 (hu5c8) has shown therapeutic effects on SLE patients in clinical trials [17,30]. Another humanized monoclonal antibody, IDEC-131, was tested in a phase II clinical study in ITP patients [31]. However, they were not approved for clinical use because of thrombotic complications when BG9588 was used in some SLE patients and IDEC-131 was used in treating Crohn's disease [27]. A third humanized anti-CD40L antibody, ABI793, targeted a different epitope and was found to have the same thrombotic complications, suggesting that these complications are a common effect of anti-CD40L antibodies regardless of epitope specificity. Recently, researchers have found that the interaction of the Fc fragment of the anti-CD40L antibody with the Fc receptor CD32 in platelets may cause platelet cross-linking and lead to clotting [32]. This is   6 CD40-N disrupts the interaction of CD40 and CD40L or CD40-specific agonist antibody in vitro. a BJAB cells were stimulated with CD40L at 24 h in the presence of titrated purified CD40-N protein (0, 0.1, 1, 10 μg/mL). Western blot was performed to examine p100 procession into p52. b BJAB cells were stimulated with G28-5 (10 μg/mL) for 1 h with or without CD40-N. Real-time PCR was performed to examine the gene expression consistent with the fact that thrombus formation was not observed in mouse models because mouse platelets do not express a homolog of CD32. Reconstruction of the Fc fragment of the antibody had been shown to eliminate the complications while maintaining the therapeutic effects of the anti-CD40L antibody in SLE and MS mouse models [33,34]. The transformed isotype of high-affinity fragment Fab' and F(ab)'2 has also been studied. All of these methods provide new insights into the effects of disrupting CD40L-CD40 interaction using a CD40L antibody.
Another strategy to disrupt this interaction is to target CD40. Some CD40 antibodies have been tested. HCD122, an antibody that can disrupt CD40L-CD40 interaction but cannot activate CD40 signaling, has been used in a clinical trial to treat CD40 + multiple myeloma because of its ADCC function [35]. Another antibody, ch5D12, has shown some curative effect in a phase II clinical study for the treatment of Crohn's disease [19].
Targeting the CD40L-CD40 interaction is an important method of immunotherapy for cancer treatment. Dacetuzumab (or SGN-40, an anti-CD40 antibody) has been used in clinical trials for treating CLL, MM and NHL [36,37].
Given the importance of the pathway activated by CD40 in research and its clinical applications, we constructed a CD40-N expression system in P. pastoris. P. pastoris was chosen because of its high production yield, expression stability, ability to secrete proteins, moderate post-translational modifications, and simple economical culture conditions. Additionally, this expression system has been used to prepare many recombinant proteins for research and clinical applications [20,38].
Recombinant CD40-N was purified from the culture medium by a combination of Sephadex G-50 size-exclusion and Q FF-Sepharose ion exchange chromatography.
The purity of the final recombinant CD40-N exceeded 90 %. ITC assay verified the interaction of CD40-N with CD40-specific antibody. The purified CD40-N showed biologically activity based on its ability to reduce CD40L-activated non-canonical NF-κB signaling pathway and inhibit TNF-alpha expression induced by CD40-specific agonist antibody in a dose-dependent manner in vitro. Importantly, CD40-N protein could significantly decrease the inflammation in DSS-induced colitis mouse model. These data reveal that we have established a reliable method for the expression and purification of CD40-N, which is functional in vitro and in vivo in interrupting the interaction of CD40 and CD40L.
Because CD40 is a glycoprotein, glycosylation may play a critical role in its structure and function. We showed that our recombinant CD40-N was glycosylated with Nlinked sugars, which was responsible for the increased molecular weight observed by SDS-PAGE (from 19.3 to 27 kDa). The predicted potential N-linked glycosylation sites of CD40-N are Asn153 and Asn180, and the exact N-linked sugars sites remain to be elucidated.

Conclusions
This work has successfully generated CD40-N recombinant protein in Pichia pastoris that can disrupt the CD40L-CD40 interaction. It may serve as a foundation for further scientific and clinical research. The protein fragment we have acquired has potential to be used in research or even treating inflammation diseases such as colitis.


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The protein domains of the Dictyostelium microprocessor that are required for correct subcellular localization and for microRNA maturation

ABSTRACT The maturation pathways of microRNAs (miRNAs) have been delineated for plants and several animals, belonging to the evolutionary supergroups of Archaeplastida and Opisthokonta, respectively. Recently, we reported the discovery of the microprocessor complex in Dictyostelium discoideum of the Amoebozoa supergroup. The complex is composed of the Dicer DrnB and the dsRBD (double-stranded RNA binding domain) containing protein RbdB. Both proteins localize at nucleoli, where they physically interact, and both are required for miRNA maturation. Here we show that the miRNA phenotype of a ΔdrnB gene deletion strain can be rescued by ectopic expression of a series of DrnB GFP fusion proteins, which consistently showed punctate perinucleolar localization in fluorescence microscopy. These punctate foci appear surprisingly stable, as they persist both disintegration of nucleoli and degradation of cellular nucleic acids. We observed that DrnB expression levels influence the number of microprocessor foci and alter RbdB accumulation. An investigation of DrnB variants revealed that its newly identified nuclear localization signal is necessary, but not sufficient for the perinucleolar localization. Biogenesis of miRNAs, which are RNA Pol II transcripts, is correlated with that localization. Besides its bidentate RNase III domains, DrnB contains only a dsRBD, which surprisingly is dispensable for miRNA maturation. This dsRBD can, however, functionally replace the homologous domain in RbdB. Based on the unique setup of the Dictyostelium microprocessor with a subcellular localization similar to plants, but a protein domain composition similar to animals, we propose a model for the evolutionary origin of RNase III proteins acting in miRNA maturation.

Introduction
MicroRNAs (miRNAs) are highly conserved small RNAs of 21-25 nt that act in endogenous gene regulation. 1 In the canonical miRNAs biogenesis pathway, 2 successive processing events take place, mediated by the ribonuclease (RNase) III proteins Dicer and/or Drosha. [2][3][4][5] Initially, primary miRNA (pri-miRNA) transcripts featuring characteristic secondary structures with a terminal loop and a bulge-rich stem are processed in the nucleus of animals by Drosha, or the Dicer-like protein DCL-1 in plants. [2][3][4][5][6][7] In plants, the resulting stem-loop structured precursor miRNAs (pre-miRNA) are further processed in the nucleus by DCL-1 to mature miRNAs. 8 In animals, this final processing step is carried out in the cytoplasm by a Dicer protein 6 after nuclear export by exportin-5. 9,10 In general, Dicer and Drosha proteins contain bidentate RNase III domains and a double-stranded RNA binding domain (dsRBD; reviewed in 11,12 ). The RNase III domains are arranged in tandem and are thought to form the catalytic center by intramolecular pseudo-dimerization. 13 Dicer proteins additionally feature several other protein domains. Of these, their helicase and PAZ domains are of particular importance, as they have been implied in measuring the distance between the 3-overhang and the terminal loop, a strict requirement for pre-miRNA cleavage at the correct position. [14][15][16][17][18][19] Conspicuously, Drosha enzymes do not feature these domains. A current model proposes that junctions between single stranded (ss) and double-stranded (ds) parts of the pri-miRNA might determine the cleavage sites of Drosha, with both structures in the lower and the upper stem critical for precise processing. 20 Additional determinants for Drosha cleavage sites were also identified in human pri-miRNAs sequences, such as basal UG or apical GUG motifs; however, those sequence patterns do not appear to be conserved between animals. 21,22 Despite the differences between animals and plants in both subcellular localization and participating enzymes in the miRNA processing, a common theme is observed: each of the RNase III enzymes requires interaction with a dedicated dsRBD-containing protein for full functionality. The nuclear complex containing Drosha and DGCR8/Pasha in animals has been dubbed the microprocessor, 23,24 and deletion of either the RNase III protein or the double stranded RNA binding protein (DsRBP) abolishes or significantly impairs the formation of pre-miRNAs. The exclusively nuclear miRNA processing in plants is carried out by DCL1 and interaction partners within so-called nuclear dicing bodies found in the periphery of, but not within nucleoli. 25 Recent data indicate that dicing bodies co-localize with Cajal-body markers, 26 where small RNAs in plants are 2'O-methylated by the Cajal-body associated methyltransferase Hen1. 27 The species Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens all belong to the Opisthokonta, one of 5 supergroups that have evolved from the last eukaryotic common ancestor (LECA). 28 Arabidopsis thaliana belongs to another supergroup, the Archaeplastida. The miRNA processing pathways and their key players thus appear to differ in these 2 supergroups, but are similar within the Opisthokonta. Adding information about the organization of miRNA maturation outside those supergroups, we recently have reported the identification of the microprocessor in the amoeba Dictyostelium discoideum, 29 a well established model organism of the Amoebozoa supergroup. In D. discoideum, the Dicer DrnB and its associated DsRBP RbdB form the microprocessor that displays similarities in localization to the plant Dicing bodies, however, the domain composition of DrnB is more comparable with that of animal Drosha proteins (Fig. 1). Curiously, the genome of the amoeba does not encode for any PAZ and helicase domain containing Dicer protein, 30 which are, with that domain composition, central enzymes in miRNA processing in both other supergroups.
Here we investigated the functional importance of the identified protein domains in both DrnB and RbdB for correct localization and miRNA maturation.

Results
N-and C-terminal GFP DrnB fusion proteins complement the drnB null phenotype DrnB has been shown to be important for the generation of mature miRNAs in Dictyostelium discoideum. 31 Tagged versions of the protein were found to localize to nuclear foci, 32 and our recent study indicated that these foci represent nucleoli. 29 To detail the functional importance of the DrnB localization in miRNA biogenesis, we generated a series of N-and C-terminal GFP fusions of DrnB with varying expression levels. These were mediated by the use of the high copy extrachromosomal plasmid pDM317 33 or the integrating plasmid pDneo2a, 32 containing strong act15 or weak act6 promoters, 34 respectively. Upon transformation of these constructs in both the wild type AX2 and the DdrnB gene deletion 35 strains, fluorescence microscopy consistently showed localization of the fusion proteins in the nucleolar foci, 29 independently of the localization of the tag, or the type of vector used (Fig. 2a). The number of these foci varied, however, from 1 to 7 between individual cell nuclei and appeared to be correlated with differing expression levels obtained from the different plasmids (Fig. 2a,b). Expression of the GFP-fusions was observed in the majority of DdrnB cells, but only in about 1% of the AX2 wildtype, indicating that DrnB levels might be under tight control in the wild type.
We next set out to investigate whether the different GFP DrnB fusion proteins (Fig. 2a,b) can rescue the phenotype of DdrnB cells, in which mature miRNAs disappear. 35 When these cells were transformed with the constructs encoding the different GFP DrnB fusions, mature miRNAs could readily be detected by Northern Blot analysis (Fig. 2c). Again, this observation was made independently of the location of the tag, and whether the fusion protein was expressed from extrachromosomal or integrating vectors. The observation that the miRNA phenotype of DdrnB could be rescued by expression of DrnB fusion proteins suggests that their localization ( Fig. 2a) corresponds to that of the endogenous protein.
Nuclear run on experiments were carried out to detail which RNA polymerase transcribes the miRNA genes. In the presence of low concentrations of a-Amanitin, miRNAs disappeared (Fig. S1). This indicates that miRNAs in D. discoideum are transcripts generated by RNA polymerase II, similar to canonical miRNAs in animals, as summarized recently. 36

DrnB localizes to the periphery of nucleoli
We recently reported the nucleolar localization of DrnB, which was concluded from its localization to regions on the nuclear periphery that are characterized by reduced DAPI staining. 29 Here, we set out to detail the specific nuclear localization, for which we employed laser scanning microscopy on paraformaldehyde (PFA) fixed cells. As no molecular marker for nucleoli in Dictyostelium was available for our purposes, we cloned mRFP-tagged fibrillarin. Upon expression in the amoeba, we observed the expected nucleolar localization of fibrillarin by fluorescence microscopy (Fig. 3a). Its fluorescence signal perfectly co-localized with the dark arcs in the nuclear periphery, which can be observed in PFA fixed cells by phase contrast microscopy. This data confirms the hypothesis that these dark arcs are the nucleoli, as has been suggested earlier. 37 In line with our recent report for GFP-DrnB, 29 also all other GFP DrnB fusion proteins were found to localize at these arcs, as shown for the example of DdrnB:DrnB-GFP cells (Fig. 3b). A closer inspection of the nucleolar localization of GFP-DrnB suggested, however, that the signals overlap, but do not perfectly coincide (Fig. 3b). To detail this, Z-stacks through a single nucleus were recorded (Fig. 3c). These images revealed an association of the fusion protein exclusively to the periphery of nucleoli, with no observable fluorescence from within these sub-nuclear bodies. This indicates that DrnB is a perinucleolar protein.

DrnB foci do not depend of the integrity of nucleoli
Having shown that GFP DrnB fusions localize in perinucleolar foci, we were interested to study whether this localization depends of intact nucleoli. To test this question, we employed Actinomycin D (AMD), which has been shown to cause disintegration of nucleoli in Dictyostelium at concentrations that specifically inhibit RNA polymerase I 37,38 . When we treated living cells with such AMD concentrations, the DrnB foci remained (Fig. 4a), while the dark arcs representing the nucleoli (Fig. 3a) vanished. This indicates that the clustering of DrnB in the focal structures does not depend on intact nucleoli. Since nucleoli are the sites of rDNA and rRNA transcription 39 we considered that the punctate localization of DrnB might require RNA or DNA as a scaffold. To test these possibilities, we digested nucleic acids in situ with RNase A or DNase I, either individually or together, and monitored nucleic acids with Draq5 TM , which stains both RNA and DNA in Dictyostelium nuclei (Fig. 4b). When this treatment was applied to living cells, the DrnB foci remained intact (Fig. 4b). While these experiments cannot rule out that the assembly of DrnB in the perinucleolar foci might require intact nucleoli, or the presence of nucleic acids, the Localization is shown for N-and C-terminal fusion proteins expressed from integrating (int) and low copy vector pDneo2a, or extrachromosomal (ex) and high copy number pDM317 and pDM323 plasmids. Nuclei are counter stained with DAPI and the size bar represents 5 mm. (b) Western blot analyses of GFP DrnB fusions. Expression of GFP fusions of DrnB (189 kDa) were analyzed using an a-GFP antibody, and using an unrelated GFP control fusion protein of 170 kDa in size. No signal was observed for the transformed AX2 strain, where GFP-DrnB was expressed in less than 1 % of the subcloned culture, as judged by fluorescence microscopy. Expression in the DdrnB background led to detectable Western Blot signals, consistent with a higher fraction (»60%) of the transformed and subcloned cells displaying GFP fluorescence. Experiments were carried out with cells under G418 (10 mg/l) selection. (c) Northern blot analyses of ddi-miR-1177 in DdrnB cells expressing Nor C-terminal GFP DrnB from integrating (int) or extrachromosomal (ex) plasmids, compared to AX2 wild type or untransformed DdrnB cells. Ethidium bromide stained rRNA (bottom) served as loading control. data indicate that once the punctate structures are formed, they do not require intact nucleoli, or the presence of nucleic acids.

The dsRBD of DrnB is dispensable for miRNA biogenesis
We next set out to explore the functional importance of the individual protein domains of DrnB for subcellular localization and miRNA biogenesis. At the N-terminus, DrnB features a dsRBD and, toward the C-terminus, 2 RNase III domains (Fig. 5a): the same domains are found also in the nuclear Drosha proteins in animals, albeit with a shuffled order 40 . Additionally to those protein domains, using PSORT 41 , we identified a typical nuclear localization signal (NLS) consisting of SIRKRRRKR at amino acid position 493-501 (Fig. 5a). We generated a series of constructs that allowed us to overexpress protein variants, in which individual protein segments of DrnB were deleted or joined, and tested these constructs for subcellular localization and miRNA rescue in the DdrnB background (Fig. 5b).
Removal of the NLS signal resulted in the loss of the punctate foci seen for full-length DrnB, and GFP-signals were observable all over the cell; although the DNLS fusion protein was still slightly accumulated in the nucleus, the miRNA phenotype of the DdrnB cells was no longer rescued (Fig. 5). Previous studies on human Drosha have shown that its dsRBD is essential for processing of pri-miRNAs 42 leading us to assume a similar indispensable role for the dsRBD in the Dictyostelium DrnB protein. To our surprise deletion of the dsRBD containing N-terminus of DrnB did not affect complementation of the miRNA phenotype nor did it greatly alter the cellular localization of the resulting DdsRBD protein variant, as the GFP signal is also found in foci, although of smaller size (Fig. 5). This finding indicates that the dsRBD of DrnB is not required for the generation of mature miRNAs. Expression of the tandem RNase III domains alone resulted in ubiquitous distribution of the RNase III construct throughout the cell with enrichment in the nucleus, indicating that these domains might contain a noncanonical NLS (Fig. 5). This is supported, albeit indirectly, by a similar localization seen for the NLS-RNase III construct expressing  the NLS fused to the tandem RNase III domains (Fig. 5). Although both, RNase III and NLS-RNaseIII were still present in the nucleus, neither of the constructs could revert the miRNA phenotype of DdrnB cells and neither appeared accumulated in the perinucleolar foci. In summary, these data indicate that the identified NLS is required, but not sufficient for the punctate perinucleolar localization of DrnB. This localization to perinucleolar foci appears to be correlated with a function of DrnB in miRNA biogenesis, for which DrnB's dsRBD, however, is dispensable.

RbdB accumulation in perinucleolar foci depends on DrnB levels
We have recently shown that RbdB, a protein solely featuring an Nterminal dsRBD, co-localizes with DrnB. Furthermore, DrbdB cells display the same molecular phenotype in miRNA biogenesis as DdrnB cells. 29 In that study, we have also shown that the localization of the 2 proteins in the nucleolar foci is independent of each other. For the microprocessor complex in animals, a recent study has shown crossregulation of the levels of Drosha and its DsRBP DGCR8. 43 When we expressed GFP-DrnB in DdrnB cells either from a low or from a high copy vector, we observe increased numbers of DrnB foci in the nucleus (Fig. 6). Notably, this was observed despite the fact that RbdB-mRFP was co-expressed in either case from the same plasmid, indicating that its steady state levels seemed to follow that of its interaction partner DrnB (Fig. 6).
The two dsRBDs within the Dictyostelium microprocessor are functionally exchangeable We noticed that the dsRBDs of the 2 proteins, which in both cases cover the amino acid residues 9-75 in Pfam, 44 display significant sequence conservation with 92.4% identical or similar amino acids within the typical abbba fold of dsRBDs 45,46 (Fig. 7a). This high sequence identity of the dsRBDs of DrnB and RbdB prompted us to investigate whether they are interchangeable. To address this, we generated chimeric proteins, in which the endogenous dsRBD of RbdB was replaced by that of DrnB, and vice versa. Since the dsRBD of DrnB is not required for miRNA maturation in its native context (Fig. 5), it was not surprising that also the chimeric protein DrnB [dsRBD RbdB ] rescued the miRNA phenotype of DdrnB cells (Fig. S2). The other chimeric protein RbdB [dsRBD DrnB ], however can fully rescue the miRNA phenotype of DrbdB cells (Fig. 7b). These data indicate that the DrnB dsRBD is functional in the context of RbdB, albeit being dispensable for DrnB.

Discussion
All GFP DrnB full length fusion proteins studied here were capable of reverting the miRNA processing defect of the drnB deletion strain, 31 indicating that their observed localization in perinucleolar foci corresponds to that of the endogenous protein (Figs 2, 3). To our knowledge, such perinucleolar bodies have not yet been described for Dictyostelium, and they appear to not co-localize to putative Cajal body markers or gem body markers in the amoeba (Fig. S3). We show that they are stable after cellular digestion of nucleic acids, and are retained after the disintegration of nucleoli (Fig. 4). DrnB localization clearly depends on the presence of the identified NLS (residues 493-501), as deletion of this sequence results in disintegration of the foci, which is concurrent with the mutant miRNA phenotype (Fig. 5). Cellular localization signals in Dictyostelium have been analyzed before, 47 and it appears that the newly identified signal sequence is necessary for proper perinucleolar localization of DrnB. The NLS is, however, not a sufficient perinucleolar localization signal, since typical DrnB foci were not restored in cells expressing a fusion of the NLS with the RNase III domains (Fig. 5). The observation that both RNase III and NLS-RNase III localize within the nucleus but are  not functional in generation of mature miRNAs indicates a strong connection between specific perinucleolar localization of DrnB and its function in miRNA maturation.
The dsRBD of DrnB does not appear to influence the subcellular localization, and in general is dispensable for miRNA processing (Fig. 5). This observation was particularly surprising as previous studies on human Drosha have shown that its own dsRBD is essential for miRNA processing. 42 Further, with the exception of the Giardia intestinalis Dicer, which has lost its dsRBD, 48 eukaryotic Dicer and Drosha proteins generally contain this domain, as summarized recently. 11,12 Contrary to what their name suggests, dsRBDs are not necessarily involved in the binding of perfectly double-stranded RNA, 49,50 but are, as parts of Drosha and Dicer, essential for precise processing of miRNA precursors. 51 The interaction partners of eukaryotic Dicer and Drosha proteins are typically DsRBPs, 11 and we have recently identified RbdB as the cognate interaction partner of DrnB, which displays the same localization and phenotype upon deletion. 29 While we have shown in that study a physical interaction of these proteins, we could show here additionally a DrnB dependent number of foci (Fig. 6). This might point toward a genetic interaction between the 2 proteins, additionally to the previously shown physical interaction. 29 Such interactions have previously been reported also for the microprocessor complex in animals, in which Drosha regulates the expression level of its DsRBP interaction partner. 43 The highly conserved N-terminal dsRBDs of DrnB and RbdB can be functionally exchanged (Figs 7 and S2), and this implies that the dsRBD of DrnB is a functional protein domain, raising the questions of why it is not used, and why it is not lost. While experimental answers are lacking at present, it is conceivable that by outsourcing the dsRBD functionality to RbdB, DrnB attains more flexibility in additional processes that it might be involved in and, in such presumed processes, it might make use of its own dsRBD. In this context, it is worth noting that rRNA processing appears altered in DdrnB cells (Kruse and Hammann, unpublished). This lends support to the idea of additional possible cellular functions of DrnB, and similar functions have also been implied for a Dicer protein from Candida albicans. 52 Furthermore, a recent study identified a large number of Dicer-bound RNA sequences beyond the miRNA pathways in human and C. elegans cells. 53 During evolution, distinct solutions were found for the implementation of the nuclear miRNA processing. Plants feature Dicing-or Cajal-bodies, which were shown to accommodate several components of the si-and miRNA pathway such as DCL, dsRBPs, Argonautes and Hen1, 8,26,[54][55][56] and the organization of the Dictyostelium microprocessor seems similar to that observed in plants ( Fig. 3 and 29 ). In terms of the domain structures of the involved RNase III proteins, however, clear differences can be discerned, as plants employ DCL1, a genuine Dicer protein, while DrnB features the protein domains of Drosha, albeit in a shuffled version. Drosha proteins act in nuclear complexes in the RNA regulation and miRNA processing of opisthokonts, 2,23,24,51,57 which include C. elegans, D. melanogaster or mammals. Droshas have been suggested to have evolved as an independent protein family in animal lineages, based on the observation that their polypeptides outgroup with respect to plant and animal Dicer proteins. 58 The Dictyostelium RNase III proteins DrnA and DrnB feature a shuffled Drosha domain structure (Fig. 1), but cluster with the Dicer proteins. 58 The Amoebozoa, including Dictyostelium, branched off during evolution after the plants (Archaeplastida) and form a sister group of the Opisthokonta. 28 It thus seems plausible that Dictyostelium may have retained the nuclear organization of the plant-like miRNA processing pathway. This would imply that their Dicers have lost those protein domains, which are absent in the animals' Drosha proteins. This scenario of convergent evolution with respect to the Ophisthokonts, would explain the unique setting of miRNA processing in this amoeba, and beyond: also the other social amoebae encode exclusively RNase III proteins with a domain structure like DrnB (G. Gl€ ockner, personal communication), indicating this to be a general characteristic of the Amoebozoa supergroup.
In LECA, the last eukaryotic common ancestor, one Argonaute-like protein, one Piwi-like protein, one RNA dependent RNA polymerase and one Dicer were suggested to have constituted the basic RNAi machinery. 58 Based on this, we suggest a model for the evolution of RNase III proteins (Fig. 8). Possibly, LECA featured next to a nuclear Dicer already a cytoplasmatic homolog, a scenario that would still be mirrored by contemporary plants. After the split of the Amorphea in the Opisthokonta and Amoebozoa supergroups, the Dicer proteins in the latter would have lost all but their dsRBD and RNase III domains. Alternatively, the 2 domain-poor Dicers in the Amoebozoa might be derived from another by gene duplication after domain loss. Only after the split of the Amoebozoa, the tasks of miRNA maturation were spatially separated in the Opisthokonts. They appear to be the sole organisms, in which Drosha proteins evolved to process pri-miRNA transcripts in the nucleus. After export of resulting pre-miRNA into the cytoplasm, their processing by Dicer yields mature miRNAs.

Cultivation and transformation of Dictyostelium
Dictyostelium discoideum AX2 and DdrnB (rox) 35 cells were grown axenically in HL5 medium (Formedium) at 22 C under constant light. Antibiotics against prokaryotic growth (10 mg/ml Penicillin, 10 mg/ml Streptomycin, 250 ng/ml Amphotericin, 50 mg/ml Ampicillin) were supplemented by default. Transformation of D. discoideum cells was performed by electroporation as described previously. 59 12-16 hours after transformation cells were selected by 10 mg/ml G418 or Blasticidin S in HL5 for 7-10 d. Success of transformation and protein expression was verified by Western blot analysis.

Cloning of DrnB variants and RbdB
The genomic sequence of drnB was amplified and cloned into the integrating vector pDneo2a-GFP previously. 32 Full length DrnB, DdsRBD and (NLS-) RNase III domains were re-amplified by PCR for cloning into extrachromosomally replicating vectors pDM317 and pDM323 for N-and C-terminal fusion to GFP, respectively. 33 The NLS (residues 493-501) was deleted by using the StarGate Ò Mutagenesis kit (IBA GmbH, G€ ottingen). All drnB containing vectors contained G418-resistance genes. The gene for RbdB (DDB_G0269426, dictybase.org) was amplified on genomic DNA by PCR and cloned into the extrachromosomal plasmid pDM326-C-RFP featuring a Blasticidin S resistance marker. 33 All genes were sequenced after amplification by PCR and cloning. Oligonucleotides used in this study are listed in Table S1.

Shuffling of dsRBDs
The endogenous dsRBDs of DrnB and RbdB are present at the N-terminus of each gene (residues 9-75 in both cases). The first 8 encoded amino acids are the same except for position 7, where DrnB features a leucine, and RbdB a threonine. The chimeric cDNA encoding the dsRBD of DrnB in the context of RbdB was cloned as follows: The dsRBD of DrnB was amplified from cDNA using the primers FZ009 and FZ010. The forward primer introduced the mutation L7T such that the sequence upstream of the dsRBD DrnB corresponded to that of RbdB and a BamHI restriction site was added. The reverse primer introduced a silent mutation downstream of the dsRBD DrnB coding sequence (G to C transition at position 228), yielding an XbaI recognition site. The PCR-fragment was cloned in the pGEM Ò -T Easy vector (Promega). The C-terminal part of RbdB was amplified with the primers FZ011 and DM053, adding N-terminal XbaI and C-terminal SpeI restriction recognition sites, respectively. The C-terminal rbdB fragment was then ligated into the pGEM Ò -T Easy vector adjacent to the N-terminal part of the gene encoding the dsRBD of DrnB. The chimeric cDNA was cut with BamHI and SpeI and ligated into the extrachromosomal expression vector pDM323. The gene was in frame with a C-terminal GFP-tag. The chimeric cDNA encoding the dsRBD of RbdB in the context of DrnB was cloned by amplifying the N-terminal part of RbdB including its dsRBD from cDNA using primers FZ005 and FZ006. The forward primer introduced the mutation T7L. By the reverse primer, a silent mutation was introduced downstream of the dsRBD coding region (A to T transition at position 231), yielding a HindIII site. The PCRfragment was cloned in the pGEM Ò -T Easy vector (Promega). The C-terminal part of DrnB was amplified from cDNA using the primers FZ007 and FZ008. During PCR reaction, an N-terminal HindIII and a C-terminal SpeI site were added. The C-terminal drnB fragment was ligated adjacent to the N-terminal part of the rbdB gene, mainly encompassing the dsRBD of RbdB. The chimeric cDNA was cut with BglII and SpeI and then ligated into the extrachromosomal vector pDM323.

Cloning and expression of D. discoideum Fibrillarin
The genomic sequence of fbl (gene accession number DDB_G0269878, www.Dictybase.org) was amplified by PCR using the Primer pair fbl BglII forward and fbl BcuI reverse. The product was cloned into pJET1.2 (Thermo Scientific) and sequenced. Recloning of fbl into the destination vector pDM326-mRFP was conducted by restriction digestion with BglII and BcuI and ligation by using T4-DNA Ligase (Thermo Scientific). After verification of the correct insertion by restriction digestion, the plasmid was used for transformation of AX2 cells. Laser scanning microscopy was performed on fixed cells with 4 % PFA.
Cloning and expression of TC 1 and Gemin 1 Genes coding for Telomerase Cajal body protein 1 (TCAB 1) (dictybase.org accession DDB_G0269858) and Gemin 1 (dictybase.org accession DDB_G0276357) were amplified from D. discoideum AX2 genomic DNA by using primer pairs TC1 1 N BglII forward, TC 1 N BcuI reverse and Gemin 1 C BglII forward, Gemin 1 C BcuI reverse, respectively. Shown are for species of the 3 indicated evolutionary supergroups in orange the RNase III proteins Dicer and Drosha that contemporarily act in miRNA maturation, and in gray those that are currently present, but that do not contribute to miRNA maturation. FECA/LECA denote the first and the last eukaryotic common ancestor, respectively. In this model, LECA featured at least one Dicer protein in the nucleus (orange bold) that acted in miRNA processing, and possibly an additional one in the cytoplasm (gray), which was not involved in that process. After the split of the Amorphea into Opisthokonta and Amoebozoa, either domain loss happened in both Dicers in Amoebozoa, or the proteins were derived by gene duplication after domain loss in one Dicer. In Opisthokonta only did Drosha proteins evolve and the final step of the miRNA maturation was redistributed to the cytoplasmatic Dicer protein. Note that this simplified scheme only includes those evolutionary supergroups that have been analyzed with respect to miRNA maturation.

Nuclear run on assay
Nuclei of AX2 cells were isolated by sucrose gradient centrifugation as described before 60 with modification in cell lysis. Here, 5 x 10 8 cells were resuspended in 20 ml lysis buffer (50 mM Tris pH 7, 5 mM MgCl 2 , 20 mM KCl, 10 % sucrose, 1 mM dithiothreitol, 0.2 mM phenylmethylsulphonyl chloride) and disrupted mechanically by the use of a cell homogenizer (Isobiotec, Heidelberg) with a clearance of 10 mm.
Nuclear run on reactions were carried out as described previously. 61 For inhibition of RNA Polymerase II, a-Amanitin (Applichem) was added to a concentration of 330 ng/ml for 15 minutes or 30 minutes, before addition of [ 32 P]UTP (Hartmann). RNA was extracted with 1 ml Trizol Ò (Life technologies)/0.2 ml chloroform. The aqueous phase was filtered through Sephadex G50 and RNA was denatured at 95 C prior to slot blot hybridization. For the slot blot, 100 pmol of each DNA oligonucleotide probe (Sigma Aldrich) were transferred to nitrocellulose filters by using a vacuum slot blot apparatus. Oligonucleotides were then chemically crosslinked as described 62 and hybridized with radioactively labeled run-on RNA at 42 C over night. After three washing steps with 0,1%SDS/0,1xSSC, filters were exposed to Phosphoimager plates for 24 h.

Fixation of Dictyostelium and microscopy
GFP or RFP expressing cells were grown on coverslips in 24well plates for 1-12 hours prior fixation with 4 % PFA in Soerensen phosphate buffer (2 mM Na 2 HPO 4 , 15 mM KH 2 PO 4 ; pH 6,0) for 1 hour at 22 C. Fixed cells were stored protected from light in 4 % PFA at 4 C until use. Alternatively, cells were fixed with methanol at -20 C for 20 min. After three wash steps with 1 x PBS cover slips were covered with embedding medium Mowiol 4-88/DABCO (Carl Roth GmbH) and mounted on a microscopy slide. Imaging of cells was conducted with a fluorescence microscope (Axioplan 2, Carl Zeiss Jena GmbH) or a confocal laser scanning microscope (LSM 510 Meta; Carl Zeiss Jena GmbH, Jena, Germany) and analyzed with the LSM 510 software, Release 3.2 (Carl Zeiss Jena GmbH).

Actinomycin D treatment
Live cells grown on coverslips were treated with 0.05 mg/ml Actinomycin D (1 mg/ml in DMSO) in HL5 for 4 hours at 21 C prior fixation with 4 % PFA.

RNase A and DNase I treatment of live cells
Live D. discoideum cells were permeabilized on coverslips by 3 washes with 0.05 % (v/v) Tween Ò 20 / 2.5 mM MgCl 2 in 1 x PBS. RNase A (Sigma Aldrich) and DNase I (Thermo Scientific) were added at concentrations of 0.1 mg/ml and 50 u/ml in 2.5 mM MgCl 2 / 1 x PBS, either separately or together, and incubated at 37 C for 1 hour in the dark, as described before. 63 Cells were subsequently fixed with 4 % PFA, and nucleic acids were stained with 5 mM Draq5 (Biostatus) in 1xPBS for 20 min at 21 C and visualized by excitation at 647 nm wavelength.

Northern blotting of miRNA
Small RNAs were blotted and chemically crosslinked as described previously. 62,64 For detection of miRNAs, DNA oligonucleotides were 5-radio-labeled by T4 Polynucleotide kinase and purified by Sephadex-G50 filtration. Hybridized filters were exposed to phosphor imager plates (Fujifilm) for 5-7 d. Plates were read by the Phosphoimager FLA3000 (Fujifilm, D€ usseldorf) and images analyzed with the Multigauge Software (release 3.2).

Disclosure of potential confllicts of interest
No potential conflicts of interest were disclosed.


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Aflatoxin B1 invokes apoptosis via death receptor pathway in hepatocytes

The fungal metabolites produced by Aspergillus flavus and Aspergillus parasiticus cause detrimental health effects on humans and animals. Particularly aflatoxin B1 (AFB1) is the most studied and a well-known global carcinogen, producing hepatotoxic, genotoxic and immunotoxic effects in multiple species. AFB1 is shown to provoke liver dysfunctioning by causing hepatocytes apoptosis and disturbing cellular enzymatic activities. In liver, AFB1 causes apoptosis via extrinsic mechanism because of high expression of death receptor pathway. The detailed mechanism of AFB1 induced hepatocytes apoptosis, via death receptor pathway still remains elusive. So the present study was conducted to explore apoptotic mechanism initiated by death receptors and associated genes in aflatoxin B1 induced liver apoptosis in chickens fed with AFB1 for 3 weeks. Results from the present study displayed histopathological and ultrastructural changes in liver such as hydropic degeneration, fatty vacuolar degeneration and proliferation of bile duct in hepatocytes in AFB1 group, along with imbalance between reactive oxygen species (ROS) and antioxidant defense system upon AFB1 ingestion. Moreover, AFB1 intoxicated chickens showed upregulation of death receptors FAS, TNFR1 and associated genes and downregulation of inhibitory apoptotic proteins XIAP and BCL-2. The results obtained from this novel and comprehensive study including histopathological, ultrastructural, flow cytometrical and death receptor pathway gene expression profiles, will facilitate better understanding of mechanisms and involvement of death receptor pathway in hepatocytes apoptosis induced by AFB1 and ultimately may be helpful in bringing down the toxigenic potential of AFB1.

INTRODUCTION
Aflatoxins are probably the most intensively researched mycotoxins in the world as 4.5 billion people worldwide suffer from intemperate exposure to aflatoxins which causes 4.6-28.2% of all global hepatocellular carcinoma cases [1,2]. These fungal metabolites are mainly produced by Aspergillus flavus and Aspergillus parasiticus. The most common aflatoxins are B 1 , B 2 , G 1 and G 2 which are naturally present in many food products and affect more than one organ system simultaneously, therefore producing cascade of responses in the affected organism [3,4]. Among the major aflatoxins, aflatoxin B1 (AFB 1 ) is the most widely known carcinogen [5] having highly hepatotoxic, genotoxic, immunotoxic, and other adverse health effects on humans and several other animal species [6]. International Agency for Research on Cancer (IARC) have produced sufficient evidences of carcinogenicity of AFB 1 and classified it as a Group 1 human carcinogen and intended no safe dose [7].
As the major drug metabolizing and detoxifying organ in the body, liver is primarily affected followed by ingestion of the aflatoxins [8,9]. AFB 1 is potent hepatocarcinogen in humans and exposure to AFB 1 is known to cause both chronic and acute hepatocellular injury. AFB 1 action mechanism involves DNA adducts formation and mutation at codon 249 of the p53 tumor suppressor gene leading to Research Paper carcinogenicity in humans [10][11][12]. Although AFB 1 are hepatotoxic, causing pallor discoloration, enlargement, congestion and necrosis of liver along with triggering proliferation of bile duct and infiltration of mononuclear and heterophilic cells in many livestock and laboratory animals, yet susceptibility varies with breed, species, age, dose, length of exposure and nutritional status [13][14][15].
Apoptosis is a programmed form of cell death [16,17] and many studies have demonstrated its role in implicating pathogenesis of multiple diseases in humans and animals [18][19][20]. It is also well documented in different model systems that aflatoxins react antagonistically with different cell proteins, leading to inhibition of carbohydrate and lipid metabolism and protein synthesis, which can induce apoptosis [21]. Liao et al., 2014, investigated that AFB 1 can provoke liver dysfunctioning by promoting hepatocytes apoptosis and disturbing cellular enzymatic activities. AFB 1 induced apoptosis can be initiated by two main apoptotic pathways: the extrinsic and the intrinsic. Although intrinsic pathway is also involved in different tissue apoptosis [22,23], yet in liver cells due to high level of death receptor expression, apoptosis predominantly occurs via the extrinsic pathway [24] and contributes to the development of a number of liver diseases including liver cirrhosis, cholestasis, viral hepatitis and hepatocellular carcinoma [25,26]. Our previous studies have revealed that AFB 1 negatively affect the spleen, thymus, jejunum and ilium and elucidated the mechanisms by which AFB 1 induces apoptosis in these organs and tissues in chicken [27][28][29][30][31]. Looking at the earlier reports in different organ systems, it would be enthralling to find out how liver is affected at cellular and molecular level, by AFB 1 . In order to understand this, the present study is designed to explore the apoptotic mechanisms primed by death receptor molecules in AFB 1 induced liver apoptosis by analyzing histological, ultra structural, biochemical, flow cytometrical and relative expression changes in apoptosisregulating genes in hepatocytes using chicken as a research model, considering the fact that chicks are susceptible to AFB 1 exposure [32]. As cell proliferation and apoptosis are directly related to carcinogenicity [33], the outcomes from present study could facilitate the understanding of AFB 1 induced carcinogenicity and may be helpful in bringing down the toxigenic potential of AFB 1 .

Histological and ultrastructural analysis
Microscopically, degenerative reversible lesions were seen, from mildest to severest degree with various distributions in AFB 1 group. Compared to control group ( Figure 1A), slight to moderate hydropic degeneration, fatty vacuolar degeneration and proliferation of bile duct in hepatocytes were seen in AFB 1 group. Moreover pyknotic and fragmented nuclei were also visible in AFB 1 group ( Figure 1B). Results of ultrastructural observation revealed irregular, fragmented and condensed nucleus, swollen mitochondria with reduced number of cristae and swollen endoplasm reticulum in AFB 1 group ( Figure  1D-1F) when compared to control ( Figure 1C). AFB 1 induced histopathological lesions were observed in chicken liver. The incidence of liver congestion, vacuolar or fatty degeneration, proliferation of bile duct and nuclear fragmentation were seen going up from day 7 to day 21 in AFB 1 intoxicated chickens whereas no lesions were observed in the control group (Table 1).

Apoptotic percentage by flow cytometer
FITC Annexin V was used to quantitatively determine the percentage of cells within groups that were actively undergoing apoptosis. Apoptotic cell counts were determined by examining the total percentage of early (Annexin-V positive and PI negative) and late (both Annexin-V and PI positive) apoptotic cells. Figure 2A, 2C, 2E and 2B, 2D, 2F shows scattered analysis of early and late hepatocytes apoptosis at 7, 14 and 21 days in the control and AFB 1 groups respectively. When compared to the control, the AFB 1 group at 7, 14 and 21 days displayed a significantly increased percentage of apoptotic cells (p<0.05 or p<0.01) ( Figure 2G).

Biochemical analysis
Compared to the control group, the activities of CAT (Catalase) and GSH-Px (Glutathione Peroxidase) in the AFB 1 group were observed significantly downregulated (p<0.05 or p<0.01) throughout the experiment at different time points. Activity of SOD (Superoxide Dismutase) and Hydroxyl free radical scavenging were also seen remarkably down (p<0.05 or p<0.01) in the AFB 1 group compared to the control at 14 and 21 days. The contents of MDA (Malondialdehyde) were observed significantly upregulated at 7, 14 and 21 days (p<0.01), while GSH (glutathione) contents were seen reduced (p<0.05 or p<0.01) at 14 and 21 days in AFB 1 group when compared to the control group ( Figure 3).

qRT-PCR analysis of relative expression of genes involved in death receptors induced apoptosis
The death receptor genes FAS (Fatty acid synthetase receptor) and TNF R1 (Tumor necrosis factor receptor 1), and the genes involved in DISC (death-inducing signaling complex) formation i-e FADD (FAS-associated death domain), TRADD (TNF receptor-associated death domain), and TRAF2 (TNF receptor-associated factor 2) were seen significantly upregulated (p<0.05 or p<0.01) at 7, 14, & 21 days with an exception of TRAF2 which was not significantly upregulated at day 7 in AFB 1 group when compared with the control group. The mRNA levels of CASPASE (cysteine-aspartic protease) family genes, i-e. CASPASE 3, CASPASE 8, CASPASE 9, and CASPASE 10 were seen significantly raised at 14 and 21 days (p<0.05 or p<0.01), also CASPASE 3 was seen significantly upregulated at day 7 in AFB 1 group compared to the control. BCL-2 (B-cell lymphoma 2) and XIAP (X-linked inhibitor of apoptosis protein) genes displayed notable downregulation (p<0.05 or p<0.01) at day 14 and 21, while mRNA levels Incidence of histopathological lesions in the liver among chickens from different experimental groups (n=6) www.impactjournals.com/oncotarget of IKK (IκB kinase) did not change throughout 7, 14 and 21 days, although it was seen increasing at day 21 but not significant enough (p>0.05) ( Figure 4).

DISCUSSION
In the current study we scrutinized the mechanisms of AFB 1 induced hepatocyte apoptosis by judging the involvement of apoptosis-associated genes and oxidant situation. The histopathological changes in AFB 1 intoxicated livers were in general agreement with previous reports [34,35]. Slight to moderate hydropic degeneration, fatty vacuolar degeneration, proliferation of bile duct, massive congestion in midzonal areas, pyknotic and fragmented nuclei within hepatocytes were seen in AFB 1 intoxicated chickens. Furthermore various electron microscopically  shows Hydroxyl free radical scavenging while E and F. bar graphs display contents of MDA and GSH in chicken hepatocytes (number of chickens, n = 6). *p<0.05, or **p<0.01, when compared to the control. Figure 4: Relative expression of genes involved in death receptor induced apoptosis in liver of chickens exposed to AFB 1 . Figure 4 represents the mRNA levels of FAS, TNF-α, FADD, TRADD, TRAF2, CASPASE 10, CASPASE 8, BCL-2, IKK, XIAP, CASPASE 9 and CASPASE 3 in liver of the AFB 1 -fed chickens and expressed as fold change relative to the control group (number of chickens, n = 6). *p<0.05, or **p<0.01, when compared to the control. detectable changes characterized apoptosis, such as condensed and hyperchromatic chromatin, fragmented nuclei, swollen mitochondria and swollen endoplasmic reticulum, which were in concordance with previously published work by several research groups [16,19,36]. The flow cytometric analysis revealed increased hepatocyte apoptotic percentage in AFB 1 group, which was similar to our previous findings in thymus, spleen, intestine and bursa of fabricius [23,30,31,37] and this increased hepatocyte apoptosis can possibly be associated with AFB 1 induced carcinogenicity since the rates of cell proliferation and cell death are directly related to cancer [32]. To understand the involvement of apoptosis associated genes in liver cells, we further explored the expression of death receptor signaling pathway involved in hepatocyte apoptosis.
Induction of apoptosis can be triggered by the activation of death receptors including FAS, TNF-α, followed by DISC (death-inducing signaling complex) formation, which consists of CASPASE 8, and associated proteins such as FADD, TRADD and TRAF2 [17]. The present study revealed over expression of FAS and TNF-R1 receptors along with their associated genes i-e CASPASE 8, CASPASE 10, FADD, TRADD and TRAF2, which was consistent with earlier reports in human hepatocytes [38,39] and with our recent study on thymocytes apoptosis [23]. IKK is crucial in activation of NF-kB based intracellular survival signaling and can activate the expression of several anti-apoptotic proteins [40,41]. XIAP is apoptotic inhibitor that can plug up the process of cell death induced by any foreign stimuli [42]. In our present study mRNA levels of IKK were not seen significantly increased throughout the experiment at different time points and XIAP expression was significantly decreased at 14 and 21 days and the expression of FADD was much higher than TRAF2. Taken all together present study insinuated that TNF1-TRADD mediated signaling is originating from TRADD, supported by ability of FADD to activate apoptosis rather than activating NF-kB cell survival signaling.
The CASPASE-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals [43] and overexpression of CASPASE family genes can force cells to undergo apoptosis. In present study, AFB 1 administration led to overexpression of CASPASE family genes (CASPASE 10, 8, and 3), suggesting that apoptosis induced by FAS and TNFR1 activates CASPASE 8, which contains an N-terminus with FADD, so providing a direct link between cell death receptors and the Caspases. In addition overexpression of CASPASE 9 and down-regulation of BCL-2 genes indicated that mitochondrial pathway was also involved in hepatocyte apoptosis induced by AFB 1 (Figure 5). These results were in agreement with previous studies in human and rat hepatocytes [44][45][46][47] and our recent report in thymus apoptosis induced by AFB 1 [23].
Recently many studies have focused on revealing the pivotal role of ROS as well as resulting oxidative stress in inducing apoptosis [48,49]. It's also previously been demonstrated that disequilibrium between ROS formation and antioxidant defense system may cause DNA damage and mitochondrial disintegration by producing oxidative stress which could lead to apoptosis [50][51][52]. Oxidative damage is relieved and free radicals are removed from the system by crucial catalyst, such as SOD, CAT and GSH-Px. O 2 is converted in to H 2 O 2 by SOD, which is further converted in to H 2 O by GSH-Px [53]. MDA is an important index of antioxidant ability, since it is the main product of lipid peroxidation [54]. In present study, activities of CAT, GSH-Px and SOD were observed significantly decreased in AFB 1 treated group along with hydroxyl ion free radical scavenging. A significantly increased contents of MDA and reduced contents of GSH were seen in AFB 1 treated group. These results were constant with previous studies in multiple organs in different species [23,30,50,[55][56][57]. These results clearly demonstrated that AFB 1 could cause imbalance between ROS formation and antioxidant defense by accumulating ROS and producing oxidative stress that could play an important role in mediating hepatocytes apoptosis.

MATERIALS AND METHODS
All the chickens and AFB 1 experiments have been conducted in accordance with the ethical standards, declaration of Helsinki and national and international guidelines have been approved by Sichuan Agricultural University Animal Care and Use Committee (Approval No: 2012-024).

Chickens and diets
One hundred and fifty-six one-day-old healthy Cobb-500 broilers were purchased from Chia Tai Group (Wenjiang, Sichuan, China), and were randomly divided into two groups which are the control group (0 mgAFB 1 / kg of basal diet) and AFB 1 group (0.6 mg AFB 1 /kg of basal diet) with 26 chickens in each group and each group in triplicates. The basal diet, which was control diet, was formulated according to National Research Council (NRC, 1994) [58] and Chinese Feeding Standard of Chicken (NY/ T33-2004) recommendations. The AFB 1 -contaminated diet was prepared, using the method described by Kaoud [59]. Briefly, 27 mg AFB 1 (A6636, Sigma-Aldrich, USA) was dissolved into 30 ml methanol, and the solution was mixed with 45 kg corn-soybean basal diet to formulate the AFB 1contaminated diet. The control diet was prepared using the same constituents except AFB 1 . Then the methanol of diets was evaporated at 98 °F (37 °C). The AFB 1 concentrations were analyzed by HPLC (Waters, Milford, MA, USA) with fluorescence detection (Waters, Model 2475, Milford, MA, USA), and the AFB 1 concentration was determined as <0.001 mg/kg and 0.601 mg/kg respectively in the control diet and AFB 1 diet. chickens were housed in cages with electrically heated units and provided with water as well as aforementioned diet ad libitum for 21 days.

Histopathological and ultrastructural examination
At the age of 7, 14 and 21 days, six chickens in each group were euthanized and the livers were fixed in 4% paraformaldehyde (PFA) and routinely processed in paraffin. Thin sections (5 μm) of tissue were sliced, mounted on glass slides and stained with hematoxylin and eosin Y. The histological organization of the tissues were contemplated and snapped with a digital camera (Nikon, eclipse 50i, Japan).
In each group one chick per replicate, was euthanized and then immediately necropsied at the end of the trial. Small pieces of liver tissues were immediately fixed with 2.5% glutaraldehyde and postfixed in 2% Veronal acetate-buffered OsO 4 . The tissues were embedded in Araldite after dehydrating in alcohol gradient. The blocks were sectioned in 65-75 nm thick sections in a microtome with a glass knife and placed in uncoated copper grids. The sections were stained with uranyl acetate, and post-stained with 0.2% lead citrate. The subcellular architecture of liver was examined with a Hitachi H-600 transmission electron microscope (Japan).

Annexin V apoptosis detection by flow cytometry
At 7, 14, and 21 days of the experiment, six chickens in each group were euthanized, and livers were sampled from each chick to determine the percentage of apoptotic cells by flow cytometer, using the method by Chen et al. 2011 [60]. Briefly, the dissected livers were thereupon homogenized to form a cell suspension and filtered, and then the cells were washed and resuspended in phosphate buffer at a concentration of 1x 10 6 cells/mL. 5 μL Annexin V-Fluorescein isothiocyanate (V-FITC) and 5 μL propidium iodide (PI) were added into 100 μL cell suspension, and incubated at 25 ºC for 15 min in the dark. 400 μL 1 x Annexin binding buffer was added to the mixture, and then the apoptotic cells were assayed by flow cytometer (BD FACSCalibur) within 1 h. The annexin V-FITC Kit was obtained from BD Pharmingen (USA, 556547).

Quantitative real-time PCR (qRT-PCR)
Livers from six chickens in each group were removed at 7, 14, and 21 days of age, and instantly stored in liquid nitrogen. The livers samples were homogenized in liquid nitrogen, by crushing with a mortar and pestle and the powdered tissues were collected into eppendorf tubes and stored at -80°C. Total RNA was extracted using TriPure Isolation Reagent kit (Cat No. 11667165001, Roche Applied Science, Germany) following manufacturer's protocol. The quality and quantity of total RNA was measured spectrophotometrically. Extracted RNA was forthwith reverse-transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Cat No: 04897030001, Roche Applied Science, Germany). qRT-PCR reactions were performed in a total volume of 20 μL using FAStStart Universal SYBR Green Master mix (Cat No: 04913914001, Roche Applied Science, Germany), at the following thermocycler program; Initial denaturation at 95 °C for 10 min, followed by 44 cycles of "10 s at 95 °C and 30 s at melting temperature (T m ) of a specific primer pair", and melt curve analysis by 10 s at 95 °C, and 72 °C for 10 s, using Thermal Cycler (Step One Plus, Applied BioSystems, USA). β-actin was used as an internal control [61,62]. Primers information is provided in Table 2. The qRT-PCR data were analyzed and fold change in expressions were calculated using 2 -ΔΔCt calculation method described by [63].

Biochemical analysis
Six chickens in each group were euthanized and immediately necropsied at 7, 14 and 21 days of age. Then livers were immediately taken out and chilled to 0°C in 0.85% NaCl, and then dried, weighed and homogenized in 9 vol of icecold 0.85% NaCl in a chilled homogenizer and centrifuged at 3500 × g at 4°C for 10 min. Total protein was measured by the method of Bradford [33] The commercial kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and used to detect total protein (Total protein quantification kit No.A045), activities of SOD (Superoxide dismutase detection kit No.A001-1), CAT (Catalase detection kit No.A007), GSH-Px (Glutathione peroxidase detection kit No.A005) and contents of GSH (Glutathione detection kit No.A006) and MDA (Malonaldehyde detection kit No.A003-1), and − OH radical (Hydroxyl free radical detection kit No.A018) in the supernatant, according to the manufacturer's instructions.

Statistical analysis
The significance of difference between two groups was analyzed by variance analysis, and results are expressed as the mean value with deviation. The analysis was performed using the independent sample t test of SPSS software for Mac v.20.0 (IBM Corp, Armonk, NY, USA) and a value of p<0.05 was considered significant, while p<0.01 was considered markedly significant.

CONCLUSION
Tissue specific apoptosis caused by AFB 1 induced toxicity has been studied in much detail, yet the mechanisms of death receptor induced apoptosis in hepatocytes remain least explored. In present study, we have delineated the mechanisms involved in death receptor provoked apoptosis using chicken liver as a model system. The results obtained in this study could be further used www.impactjournals.com/oncotarget to understand involvement of death receptors in apoptosis and could help in amending the toxic effect provoked by AFB 1 .

ACKNOWLEDGMENTS
This work was supported by the program for Changjiang scholars, the University Innovative Research Team (IRT 0848) and the Education Department of Sichuan Province (2012FZ0066) and (2013FZ0072).