Lytic polysaccharide monooxgygenases (LPMOs) are known to boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. For their activity, LPMOs require an electron donor for reducing the divalent copper cofactor. LPMO activities are mainly investigated with ascorbic acid as a reducing agent, but little is known about the effect of plant-derived reducing agents on LPMOs activity.Myceliophthora thermophila C1, MtLPMO9A, MtLPMO9B and MtLPMO9C, differ in their substrate preference, C1-/C4-regioselectivity and reducing agent specificity. MtLPMO9A generated C1- and C4-oxidized, MtLPMO9B C1-oxidized and MtLPMO9C C4-oxidized gluco-oligosaccharides from cellulose. The recently published MtLPMO9A oxidized, next to cellulose, xylan, β--glucan and xyloglucan. In addition, MtLPMO9C oxidized, to a minor extent, xyloglucan and β--glucan from oat spelt at the C4 position. In total, 34 reducing agents, mainly plant-derived flavonoids and lignin-building blocks, were studied for their ability to promote LPMO activity. Reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose for all three MtLPMOs. Low activities toward cellulose were observed in the presence of monophenols and sulfur-containing compounds.Here, we show that three LPMOs from the fungus Several of the most powerful LPMO reducing agents of this study serve as lignin building blocks or protective flavonoids in plant biomass. Our findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. This work strongly supports the idea that the activity of LPMOs toward lignocellulosic biomass does not only depend on the ability to degrade plant polysaccharides like cellulose, but also on their specificity toward plant-derived reducing agents in situ.The online version of this article (doi:10.1186/s13068-016-0594-y) contains supplementary material, which is available to authorized users. Aspergillus and Trichoderma strains. Alternatively, the ascomycete Myceliophthora thermophila C1 is used to produce plant polysaccharide-degrading enzymes [Plant biomass utilization is considered to be a green approach for the production of renewable biofuels and biochemicals. Here, current developments aim at the effective degradation of the plant biomass polysaccharides, mostly embedded in a lignocellulosic complex, into monosaccharides using enzyme cocktails. These commercial enzyme preparations usually originate from fungi such as  enzymes .Lignocellulosic plant biomass is composed of the aromatic heteropolymer lignin and polysaccharides, such as hemicellulose and cellulose. In this research, we focus on the degradation of the highly recalcitrant polysaccharide cellulose. Cellulose is a homogenous polymer consisting of β-1 → 4)-linked glucosyl chains. The interactions of these glucosyl chains via hydrogen bondings and van der Waal forces lead to the formation of crystalline cellulose regions -linked g) databasThe AA9 classified fungal LPMOs exhibit the oxidative cleavage of cellulose-yielding products with either the C1- (lactones) or C4-  position oxidized, or mixtures of the two , 13–15. PcGH61D did not affect the amounts of products released [M. thermophila C1 do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity [M. thermophila C1. One of these LPMOs is MtLPMO9A, which has been shown to oxidize cellulose at the C1- and C4 position [MtLPMO9B and MtLPMO9C, we also characterized the C1-/C4-regioselectivity and substrate specificity. Based on their chemical structure, all reducing agents were classified into five groups, partly reflecting their effect on the LPMO activity.Most studies on the activity of LPMOs published so far have been carried out with ascorbic acid as reducing agent , 16, 17.released . Hence, cificity . Therefoposition . For theMtLPMO9B and MtLPMO9C were produced in the homologous host M. thermophila C1 and purified from the culture broth supernatant in either three (MtLPMO9B) or four (MtLPMO9C) steps. Both final MtLPMO9B and MtLPMO9C preparations showed single bands on SDS-PAGE  calculated from the respective amino acid sequences. However, ESI-MS spectra  of MtLPMO9B and MtLPMO9C showed that both LPMOs were glycosylated by hexoses. In particular, up to 13 and 5 glycosyl units were attached to MtLPMO9B and MtLPMO9C, respectively , in both the presence and absence of ascorbic acid. Incubation of RAC in the presence and absence of ascorbic acid without LPMO addition did not cause auto-oxidation of RAC, since no non-oxidized or oxidized gluco-oligosaccharides were detected using HPAEC and MALDI-TOF MS. The products released from RAC, after incubation with MtLPMO9B or MtLPMO9C, are shown in Fig. n), only C1-oxidized gluco-oligosaccharides (GlcOSn#) were formed upon incubation of RAC with MtLPMO9B in the presence of ascorbic acid , besides non-oxidized gluco-oligosaccharides (GlcOSn)  and C1-oxidized cellohexaose appeared either as a lactone (995 Da) or as the corresponding aldonic acid (1013 Da)  of cellohexaose and non-oxidized cellohexaose  Fig. a, b. For-positon .Fig. 1AcMtLPMO9A, MtLPMO9B and MtLPMO9C were constructed, based on the sequences of a C1-, C4- and C1- and C4- oxidizing LPMO as presented by Borisova et al. . The overall sequence identity of MtLPMO9A, MtLPMO9B and MtLPMO9C ranges from 41 to 46 %. All three MtLPMOs share the LPMO typical β-sheet core, but differ in their loop regions L2 (10–49), LS (114–128) and LC (176–226) that are involved in shaping the substrate-binding surface  loop region , which is not present in MtLPMO9A and MtLPMO9C  for MtLPMO9A and Cys28-Cys178 (L2–LC) for MtLPMO9B  . Reducing agents of group V have neither a phenolic ring nor a sulfur atom.Based on the structural similarities of functional groups, the 34 reducing agents tested were classified into five groups  on the amounts of oxidized and non-oxidized gluco-oligosaccharides released from RAC incubated with MtLPMOs enabling the oxidative cleavage of RAC. Nevertheless, the LPMO activity, based on the amounts of oxidized gluco-oligosaccharides formed, varied per reducing agent , but was a less efficient electron donor for MtLPMO9B and MtLPMO9C . Some compounds bearing a 1,2-benzenediol moiety, for example quercetin or taxifolin (no. 23), and the benzenetriol tannic acid (no. 28) did not donate electrons to all three MtLPMOs. Out of seven monophenols, only sinapic acid (no. 5) acted as electron donor for all three MtLPMOs, but less efficient (4–23 %) compared to ascorbic acid (no. 33). A poor electron-donating capacity was also found for sulfur-containing compounds, such as reduced glutathione (no. 29) and l-cysteine (no. 30)  differ slightly from the predicted masses, calculated from the amino acid sequences . Both MtLPMO9B and MtLPMO9C contain multiple glycosylations that were determined as hexoses based on LC/ESI-MS  compared to the released products from RAC by the MtLPMO9A using ascorbic acid as a reducing agent -glucan from oat spelt or barley and xyloglucan from tamarind seed, at the C1 and C4 position. In addition, MtLPMO9A also oxidized xylan in the presence of cellulose [MtLPMO9B oxidized RAC at the C1 position. The presence of the CBM1 linked to MtLPMO9B is not expected to have an impact on the substrate specificity of MtLPMO9B. It has been shown that the removal of a CBM1 linked to NcLPMO9C, which has a broad substrate specificity, does not affect the ability to oxidize different polysaccharides such as cellulose, cellopentaose or xyloglucan [MtLPMO9B shows a typical insertion, which forms the L3 loop region in C4-oxidizing AA9 LPMOs. Compared to the solely C4-oxidizing NcLPMO9C, MtLPMO9B shares within this L3 loop only a low sequence identity [MtLPMO9C also contains the L3 loop and is, like NcLPMO9C, a C4-oxidizing AA9 LPMO -glucan from oat spelt and xyloglucan from tamarind seed at the C4 position  from N. crassa shares the highest sequence identity (83 %) with MtLPMO9C, but unlike MtLPMO9C it has not been tested if NcLPMO9D is active on hemicellulose such as -glucan [ellulose . MtLPMO9loglucan . Besidesidentity , 25. Theidentity . MtLPMO9)-glucan , 29.We show here that three LPMOs from the same organism differ in their reducing agent specificity. Importantly, all three LPMOs are able to utilize various natural phenolic compounds as reducing agents. Most of these reducing agents are present in plants, either free or as lignin building blocks, such as sinapic acid (no. 5), or as flavonoids such as catechin (no. 9) and dopamine (no. 20). This finding is of high relevance as these reducing agents can act as intrinsic electron donors in plant biomass biorefinery .MtLPMOs, phenolic compounds with 1,2-benzenediol and 1,2,3-benzenetriol moieties yielded the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose compared to monophenols or sulfur-containing compounds , but for MtLPMO9B this number was only two . The charge differences in the vicinity of the copper ion might contribute to the different electron donor specificities of the MtLPMOs.Remarkably, the number of reducing agents that gave an activity of 70 % or higher of the LPMO activity obtained with ascorbic acid differed between the three LPMOs Fig. ; Table 2two Fig. ; Table 2NcLPMO9C , 26. IndM. thermophila C1 have their pH optimum around pH 5.0. It can be expected that this pH does not represent the optimal condition for each reducing agent tested, since redox potentials of reducing agents are pH dependent [The incubations performed in this work have been conducted at a single time point 24 h) and at one pH (5.0). This pH plays an important role for the LPMO application, due to the fact that cellulose cleaving cocktails produced by ependent . Furtherependent , 33. We 4 h and aOur findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. The mode of action of LPMOs is usually investigated in the presence of ascorbic acid. Here, we found that several reducing agents can donate electrons to the LPMOs with a similar efficiency as the commonly used ascorbic acid. Our findings are of high interest for industrial applications as most of these reducing agents are present in plant biomass and can act as intrinsic mediators in biorefinery processes.MtLPMO9A has been previously described [MtLPMO9B  (UniProt: KX772410) and MtLPMO9C (UniProt: KX772411) was performed using a low protease/low (hemi-) cellulose producing Myceliophthora thermophila C1 strain, which has been described elsewhere [MtLPMO9B and MtLPMO9C were purified from the corresponding dialyzed enzyme preparations using an ÄKTA-Explorer preparative chromatography system .The purification and activity of escribed . The homlsewhere , 40. TheMtLPMO9B was purified in three subsequent chromatographic steps. For the first anion-exchange step, the MtLPMO9B-containing enzyme preparation was loaded on a Source 30Q column . A 20 mM potassium phosphate buffer (pH 7.8) was used to pre-equilibrate the column. Elution was performed using a linear gradient from 0 to 1 M NaCl in 20 mM potassium phosphate buffer (pH 7.8) at a flow rate of 10 mL × min−1 and monitored at 220 and 280 nm. All fractions were collected and immediately stored on ice. Peak fractions were, based on UV (280 nm), pooled and concentrated by ultrafiltration  at 4 °C. The concentrated pools were analyzed by SDS-PAGE to determine the MtLPMO9B-containing pool (expected molecular mass 30.6 kDa). After the first purification step, cation exchange chromatography purification was applied. The MtLPMO9B containing pool was subjected to a Source 30S column  for further purification (second step). A 20 mM sodium acetate buffer (pH 5.0) was used to pre-equilibrate the column. The elution was performed by using a linear gradient from 0 to 1 M NaCl in 20 mM sodium acetate (pH 5.0) at a flow rate of 5 mL × min−1. Elution was monitored at 220 and 280 nm. Fractions obtained (10 mL) were immediately stored on ice. Peak fractions were pooled, concentrated and analyzed by SDS-PAGE as described above. In a third purification step, the MtLPMO9B-containing fraction was bound to a Source 30S column  using a 10 mM sodium acetate buffer (pH 5.0) after pre-equilibration. After protein application, the column was washed with 20 column volumes of starting buffer. Elution was performed using a linear gradient from 0 to 1 M KCl in 20 mM sodium acetate (pH 5.0) at a flow rate of 5 mL × min−1 and monitored at 220 and 280 nm. Fractions (3 mL) were immediately stored on ice. Peak fractions were pooled, concentrated and analyzed by SDS-PAGE as described above.MtLPMO9C was purified in four subsequent chromatographic steps. As a first size exclusion chromatography (SEC) purification step, the MtLPMO9C-rich enzyme preparation (40 mg × mL−1) was loaded onto a self-packed Superdex TM-75 column  and eluted at 5 mL × min−1 with a 10 mM potassium phosphate buffer (pH 7.0). Fractions (5 mL) were immediately stored on ice. Peak fractions were, based on UV (280 nm), pooled and concentrated by ultrafiltration as described above. The concentrated pools were analyzed by SDS-PAGE to determine the MtLPMO9C-containing pool (expected molecular mass 23.5 kDa). For the second SEC purification step, the MtLPMO9C-containing pool was loaded again on the Superdex TM-75 column under the same conditions. Fractions (5 mL) were immediately stored on ice. Peak fractions were pooled, concentrated and analyzed by SDS-PAGE to determine the MtLPMO9C-containing pool as described above. The MtLPMO9C-containing pool was dialyzed against a 20 mM Tris–HCl buffer (pH 8.4) using ultrafiltration . The dialyzed MtLPMO9C fraction was, for the third purification step, subjected to a Resource Q column , which was pre-equilibrated in 20 mM Tris–HCl buffer (pH 8.4) (third step). Elution was performed with a linear gradient from 0 to 1 M NaCl in 20 mM Tris–HCl (pH 8.4) over 20 column volumes at 6 mL × min−1. Fractions (3 mL) were immediately stored on ice. Peak fractions were pooled, concentrated and analyzed by SDS-PAGE to determine the MtLPMO9C-containing pool as described above. The MtLPMO9C-containing fraction was dialyzed against a 20 mM potassium phosphate buffer (pH 7.0) using ultrafiltration  and subjected to a Resource Q column . The column was equilibrated using a 20 mM potassium phosphate buffer (pH 7.0) and elution was performed using a linear gradient over 20 column volumes at 6 mL × min−1. Fractions (3 mL) were immediately stored on ice. Peak fractions were pooled, concentrated and analyzed by SDS-PAGE as described above.MtLPMO9B and MtLPMO9C was determined as described previously using a BCA Protein Assay Kit [MtLPMO9B and MtLPMO9C fractions were analyzed by LC–mass spectrometry confirming the presence of the two LPMOs by ‘The Scripps Research Institute’ .The protein content of ssay Kit . Furtherssay Kit . Pure MtMtLPMO9B and MtLPMO9C preparations (2.5 mg mL−1 in 0.1 % (v/v) trifluoroacetic acid) were analyzed using a liquid chromatography/electron spray ionization-mass spectrometry (LC/ESI-MS) as described previously [Purified Reducing agents were supplied by Sigma-Aldrich , unless stated otherwise. Taxifolin was purchased from Extrasynthese , catechol hydrate and chlorogenic acid from Thermo Fisher Scientific , tannic acid from BDH Chemical Ltd.  and naringin from Fluka Chemie .OSX, BiWX, Avicel PH-101, xylo-oligosaccharides (DP1-5) and β-(1 → 4)-linked gluco-oligosaccharides (DP1-5) were supplied by Sigma-Aldrich. WAX and β--glucan from barley and oat spelt (both medium viscosity) were purchased from Megazyme . Xyloglucan from tamarind seed was obtained from Dainippon Sumitomo Pharma . Regenerated amorphous cellulose (RAC) was prepared from Avicel PH-101 as described , 41. Glu−1, with or without addition of reducing agents listed in Table MtLPMO9A, MtLPMO9B or MtLPMO9C was added  and incubated for 24 h at 50 °C in a head-over-tail Stuart rotator in portions of 1 mL total volume  at 20 rpm. Supernatants of all incubations with and without reducing agent in the presence of LPMOs and of substrates incubated with and without reducing agents in the absence of LPMOs were analyzed by HPAEC and MALDI-TOF MS. The enzyme reactions were stopped by storing samples at −24 °C. All following sample treatments were performed on ice.Substrates (see figure captions) were dissolved in 50 mM ammonium acetate buffer (pH 5.0) to a concentration of 1–2 mg × mLMtLPMO9B was made using the available structure of NcLPMO9C from Neurospora crassa [MtLPMO9B against the Protein Data Bank (41 % amino acid identity). MtLPMO9C was, like MtLPMO9B, generated based on NcLPMO9C [MtLPMO9A, MtLPMO9B and MtLPMO9C, respectively, were calculated using H++-server with default settings [The structural model of a crassa   of the column with the starting conditions. The assignment of the C1- and C4-oxidized gluco-oligosaccharides using HPAEC is based on previous publications [MtLPMO9A, MtLPMO9B or MtLPMO9C. Standard deviations are represented  with pulsed amperometric detection (PAD) using an HPAEC system  as described previously . The temications , 15, 17,MtLPMO9B or MtLPMO9C was performed using matrix-assisted laser desorption ionization-time of flight mass spectrometry  as described previously [MtLPMO9B or MtLPMO9C, respectively, were determined and assigned as described previously [The analysis of substrates incubated with either eviously . Masses eviously .


Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. We demonstrate the expression of CDX2 and HNF4α in human biopsy samples. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2 induced expression of Tff3, villin, K8 and E-cadherin. HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett's metaplasia and are consistent with an origin of Barrett's metaplasia from the oesophageal epithelium.Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two transcription factors, Cdx2 and HNF4α in the conversion using primary organ cultures. Biopsy samples from cases of human Barrett's metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this,  BM is fCDX) 1 and 2 are involved in the initiation of BM , a nuclear receptor type transcription factor, may also be considered as a candidate for the initiation of BM. During early development of the gut, pancreas . Importaed in BM . Normal ed in BM . TherefoCDX2 and HNF4α in BM we developed a long-term adult mouse oesophageal explant model. Long-term culture of oesophageal epithelium has proven difficult. Broadly there are two sources of cells available to study BM: immortalized cell lines such as Het-1A, or ex vivo primary cell culture involving either mechanical tissue mincing or enzymatic digestion of oesophageal tissue. Cells or explants have been cultured on a variety of substrates, matrices and scaffolding, including organotypic models with multi-layered squamous cells -, K4 - and loricrin (the major component of the cornified cell envelope)- expressing cells were all present in the cultures B iv-xii.2.4vs Bi). To test the response to calcium, we cultured oesophageal explants for 5 days in 0.03 mM calcium followed by 3 days at a final concentration of 1 mM calcium  and normal (BME) calcium concentrations, to induce intestinal gene expression    B as for ey test) . It was Cdx2 in the low calcium medium, the levels of induction of Mucin2 and Villin RNA were only just detectable , Villin, Lactase, Trefoil factor 3 (Tff3), Alkaline phosphatase 1(ALP1), or Cryptdin 1  indicating that HNF4α suppresses the SSQE phenotype  , E-cadhedetected F.We also tested the effects of Cdx2 and HNF4α in combination but conditions could not be found in which the cultures remained viable.2.7HNF1α, was included. HNF1α plays a crucial role in intestinal development so we wished to determine if co-expression with HNF4α and Cdx2 could further enhance intestinal gene expression. Het1A cells were infected with virus encoding Cdx2, HNF4α and HNF1α alone or in combination and analysed by RT-PCR for the induction of Mucin2, K20, SI and Villin  but this was not significantly increased by addition of Cdx2 or HNF1α. There was no significant increase in villin expression in cells infected with the virus combinations tested . Expression of HNF4α protein in the Het-1A-HNF4α c1 clone was confirmed by immunofluorescence B and inds tested .3in vitro culture make it necessary to use an animal model for experimental purposes. We have developed an adult explant culture model that recapitulates the full repertoire of cell types found in the oesophagus . The presence of a myofibroblast connective tissue layer beneath the basal cells allows for epithelial-mesenchymal interactions and might help to maintain the oesophageal phenotype in culture and account for the model's long-term viability. Above the connective tissue layer, the K14-positive basal cells differentiated and expressed the markers involucrin and K4, prior to the formation of the cornified cell envelope in fully mature squames.. Moreov squames. Loricrinal model . LoricriWe examined the role of calcium in the differentiation of SSQE. Calcium is an essential determinant of epidermal keratinocyte proliferation and differentiation . Mouse eCdx2-expressing cells. Although at first sight a surprising result, it is consistent with other recent studies. Immortalized oesophageal cells require overexpression of the cell-cycle regulator cyclin D1 along with demethylating agents before ectopic Cdx2 expression can provoke the expression of intestinal genes   . HNF4α nlar gaps . The indhe crypt .Tff3 was expressed, we did not observe expression of Muc2 with HNF4α. However it is noteworthy that Muc2 expression is minimally altered in Hnf4α null intestine . The question therefore arises whether wn in BM , and we nic life . Further4All experiments were repeated at least three times.4.1Formalin-fixed wax-embedded sections of archival biopsy forceps specimens of human oesophagus , gastro-oesophageal junction (GOJ), stomach, small intestine and colon were obtained from the Pathology unit at the Royal United Hospital Bath (REC number: 13/YH/0197). Immunohistochemical staining for CDX2 (1:80) and HNF4α (1:80) was carried out using a polymer detection system and DAB label. Briefly, for Cdx2, tissue sections were dewaxed, rehydrated, submerged in a low pH solution , microwaved until boiling and for two minutes thereafter. Sections were heated for a further 15 min in a 99 °C waterbath, allowed to cool for 20 min, transferred to PBS and sequentially treated with 3% peroxide block and BioGenex Power Block for 10 min each. Cdx2 antibody  was diluted 1:80 (BioGenex Enhanced Antibody Diluent) and incubated with the sections for 30 min. Sections were rinsed thoroughly in PBS and treated with the Super Enhancer and Polymer-HRP reagent for 20 and 30 min respectively. Antibody detection was carried out in the presence of DAB (10 min). For HNF4α, sections were submerged in Dako EnVision™ Flex Target Retrieval Solution (high pH - diluted according to manufacturer's instructions), microwaved until boiling and microwave simmered for a further 20 min. Slides were allowed to cool for 20 min and transferred to PBS. Sections were rinsed in PBS and blocked for 2 h in 2% Roche blocking buffer followed by sequential treatments with the BioGenex peroxide and blocking solution as above. Hereafter, sections were subjected to the same protocol of antigen labelling and detection as for Cdx2 with HNF4α  also being diluted 1:80. Sections were counterstained with Gill's haematoxylin (Vector Laboratories).4.2L-glutamine . The oesophagus was cut at the proximal and distal ends to ensure that gastric and buccal mucosa were excluded. It was opened longitudinally and the epithelium stripped from the underlying connective tissue. Each sample of oesophageal epithelium was dissected into approximate 1 mm2 sections and the samples from different individual mice were cultured separately. Sections (10−15) of epithelial tissue were then inserted into furrows that had been etched onto plastic coverslips. The coverslips were then placed in a 35 mm tissue culture dish and covered with 1.5 ml of Basal Medium Eagle (BME) with Earle's salts  supplemented with 20% foetal bovine serum, penicillin/streptomycin  and 2 mM L-glutamine.All animal experiments were performed in accordance with UK Home Office regulations. Oesophagi were removed from adult CD1 mice following cervical dislocation and dissected in Minimum Essential Medium Eagle (MEM) with Hank's salts supplemented with 10% FBS , penicillin/streptomycin  and 2 mM L-glutamine (6 mM), human epidermal growth factor (5 ng/ml), ethanolamine (6.1 μg/ml), α-phosphoethanolamine (14.1 μg/ml), hydrocortisone (0.5 μg/ml) and bovine insulin (5 μg/ml) .For culturing oesophageal explants under low calcium conditions, MCDB 153  medium was supplemented with 4.3L-glutamine (Sigma) and penicillin/streptomycin . Culture medium was replaced every 2 days, and cells were subcultured (1:10) every 5–7 days.Het-1A cells  were maintained in Basal Medium Eagle medium (BME) (Sigma) supplemented with 10% (v/v) foetal bovine serum (Gibco), 2 mM Stable HNF4α expressing Het-1A cells were generated through lentiviral infection with pL-S-Hnf4α-I-EGFP28. Briefly, the lentivirus was prepared by transfecting pL-S-Hnf4α-I-EGFP and the packaging constructs pVSV-G, pREV, pGal/Pol/PRE into HEK293T cells . Virus containing medium was harvested 48 h after transfection, diluted in complete BME medium supplemented with dextran (5 µg/ml) and added to 6.9×104 Het-1A cells for 24 h. Medium was changed every 2 days following infection. Cells were split and seeded onto 96-well plates for single cell colony selection with HNF4α expression being validated by immunofluorescence and RT-PCR.4.4Fixation and immunostaining of explant cultures or adult mouse oesophageal sections was performed as described previously . Primary4.5Herpes simplex and one without. The VP16 virus was fused to the 5′ end of the full length mouse Cdx2 cDNA . Cdx2 was subcloned into a VP16-containing plasmid by ClaI digestion. The AdEasy expression system (Stratagene) was used for adenovirus delivery into cells and explant cultures. Briefly, BglII and XhoI were used for subcloning Cdx2 and VP16-Cdx2 into the pShuttle-IRES-hrGFP construct. The resulting shuttle vectors were then linearized with PmeI and cotransformed into BJ5183 electrocompetent cells with pAdEasy-1, the supercoiled viral DNA plasmid. Recombination was identified by restriction enzyme digestion analysis. The recombinant constructs were then produced in bulk in XL-10 Gold cells. Purified recombinant adenovirus plasmid DNA was digested with PacI to expose its inverted terminal repeat (ITR), and then used to transfect HEK239 cells where deleted viral assembly genes were complemented in vivo.Two viruses were constructed: one with the VP16 transactivation domain from 4.6Ad-null, Ad-RSV-GFP, Ad-CMV-Cdx2-hrGFP, Ad-CMV-VP16-Cdx2-hrGFP, Ad-CMV-HNF1α and Ad-CMV-HNF4α. for 12 h. Explants were processed for RT-PCR or immunohistochemistry up to 7 days post-infection.Each explant culture was incubated with 5×10Ad-null, Ad-CMV-HNF4α, Ad-CMV-VP16-Cdx2-IRES-hrGFP and Ad-HNF1α alone or in combination (as indicated) to an MOI of 15 in the presence of dextran (5 µg/ml) for 24 h. Cells were harvested for analysis 4 days post infection.Het-1A cells were exposed to 4.7RNA extraction, 1st strand cDNA synthesis and reverse transcription polymerase chain reaction was performed as described previously. AnnealinThe authors disclose no conflict of interest.BC, JMWS, DT. Acquisition of data: BC, LG, YC, ZDB, WYY. Analysis and interpretation of data: BC, LG, ZDB, YC, JMWS, DT. Drafting of the manuscript: BC, LG, ZDB, JMWS, DT. Critical revision of the manuscript for important intellectual content: RJ, MB, JMQ, JMF, SGW, JMWS. Material support: WYY, RJ, MB, LB. Study supervision: JMF, JMWS, SGW, DT.Study concept and design: This work was funded by Cancer Research UK, Medical Research Council and the UK India Education and Research Initiative.To date the molecular mechanisms underlying Barrett's oesophagus remain unidentified. We provide evidence for a role of the transcription factor HNF4a in the switch from stratified squamous to columnar epithelium.


AFB1 degradation by microorganisms or microbial enzymes provides a promising preventive measure. To this end, the present study tested 43 bacterial isolates collected from maize, rice, and soil samples for AFB1-reducing activity. The higher activity was detected in isolate L7, which was identified as Bacillus shackletonii. L7 reduced AFB1, AFB2, and AFM1 levels by 92.1%, 84.1%, and 90.4%, respectively, after 72 h at 37 °C. The L7 culture supernatant degraded more AFB1 than viable cells and cell extracts; and the degradation activity was reduced from 77.9% to 15.3% in the presence of proteinase K and sodium dodecyl sulphate. A thermostable enzyme purified from the boiled supernatant was designated as Bacillus aflatoxin-degrading enzyme (BADE). An overall 9.55-fold purification of BADE with a recovery of 39.92% and an activity of 3.85 × 103 U·mg−1 was obtained using chromatography on DEAE-Sepharose. BADE had an estimated molecular mass of 22 kDa and exhibited the highest activity at 70 °C and pH 8.0, which was enhanced by Cu2+ and inhibited by Zn2+, Mn2+, Mg2+, and Li+. BADE is the major protein involved in AFB1 detoxification. This is the first report of a BADE isolated from B. shackletonii, which has potential applications in the detoxification of aflatoxins during food and feed processing.Food and feed contamination by aflatoxin (AF)B Aspergillus species, mainly A. flavus and A. parasiticus [1, the most abundantly produced isotype, is highly toxic [1 to AFM1, which is detected in the milk of dairy cows that consume AFB1-infected feeds [Aflatoxins (AFs) are secondary metabolites produced by asiticus , that arasiticus ,3,4. AFBly toxic ,6. Cytoced feeds ,8.1 biodegradation by fungi and bacteria or their secondary metabolites or enzymes has been widely reported using Nocardia corynebacterioides (formerly Lavobacterium aurantiacum) [Armillariella tabescens [Pleurotus ostreatus [Bacillus licheniformis [Mycobacterium fluoranthenivorans [Rhodococcus erythropolis [Stenotrophomonas maltophilia [Myxococcus fulvus [Bacillus subtilis ANSB060 [Methods for decontamination and reduction of AFs in food sources have been widely investigated. AF inactivation by physical and chemical means is not highly effective or economically feasible ; a promintiacum) ,10, Armiabescens ,12, Pleustreatus , Bacilluniformis , Mycobacnivorans ,16, Rhodhropolis ,18, Stentophilia , Myxococs fulvus , and Bac ANSB060 . This ap ANSB060 .A. tabescens [Phanerochaete sordida YK-624 [1-degrading ability. It was also reported that a recombinant Trametes versicolor laccase enzyme expressed in Aspergillus niger degrades AFB1 [Mycobacterium smegmatis enzymes belonging to two F420H2-dependent reductase families were found to catalyze the reduction of the α, β-unsaturated ester moiety of AFs by spontaneous hydrolysis [AF-degrading enzymes have been isolated from a variety of microorganisms. Recently, an aflatoxin oxidase from abescens  and manga YK-624  were shodes AFB1 . Nine My1-degrading microbes from soils and contaminated kernels using coumarin medium. Several new AFB1-degrading bacterial strains were thus identified; among them, Bacillus shackletonii strain L7 showed the strongest activity. We evaluated the degradation efficiency of strain L7 against various AFs and purified and characterized a thermostable enzyme named Bacillus aflatoxin-degrading enzyme (BADE) responsible for AFB1 degradation activity. We also analyzed the genotoxicity of AFB1 degradation products treated with proteins from strain L7.To this end, a screening method was developed in the present study to isolate AFB1 to varying degrees in nutrient broth (NB) after incubation for 72 h at 37 °C  showed more than 70% AFB1 degradation in the medium, with the highest value (71.7%) observed for isolate L7.In total, 43 single-colony bacterial isolates were obtained from 247 samples collected from different sources, all of which were able to reduce AFBat 37 °C . Eight oBacillus shackletonii strain LMG 18435 (99% sequence similarity). This is the first report of a bacterium of this genus exhibiting mycotoxin-degrading ability. The partial 16S rRNA gene sequence of L7 was submitted to GenBank (access. no. KX364157), and the strain was deposited at the China General Microbiological Culture Collection Center (CGMCC8868).Isolate L7 was a rod-shaped Gram-positive bacterium that could grow under extreme conditions (55 °C and pH 9.0). It utilized simple sugars like glucose, maltose, and sucrose as a sole carbon source, and hydrolyzed casein but not gelatin, amylum, butyrin, or Tween 80 . Based o1, AFB2, AFG1, AFG2, and AFM1 was 92.1%, 84.1%, 63.6%, 76.1%, and 90.4%, respectively, when cultured in NB at 37 °C for 72 h  (1-degrading ability of the supernatant declined to 52.6% and 15.3% upon treatment with proteinase K without and with sodium dodecyl sulphate (SDS), respectively (1 degradation.The culture supernatant of isolate L7 was more effective than viable cells and cell extracts in degrading higher concentrations of AFB < 0.05) . The AFBectively . These r1 degradation by the culture supernatant of isolate L7 proceeded relatively rapidly and continuously, with 40.9, 77.9, and 90.3% reduction observed in the first 12 h and after 72 h and 5 days, respectively (AFBectively .p < 0.05). These results indicate that AFB1 treated with the culture supernatant of isolate L7 had no genotoxicity in the presence of the S9 crude enzyme preparation.The genotoxicity of the samples was evaluated using the SOS Chromotest, with the results expressed as induction factor ± standard deviation . The ind1-degrading ability of the culture supernatant after incubation for 24 h increased from 47.6% to 70.1% upon concentration by 50-fold by ultrafiltration and was positively correlated with protein concentration , concentrated by ultrafiltration with a cut-off molecular weight of 3 kDa, and subjected to diethylaminoethanol (DEAE)-sepharose ion-exchange chromatography. The maximum absorption of BADE was observed between 240 and 300 nm. The protein concentration of each fraction was monitored at 280 nm during enzyme purification. The separation profile of the enzyme showed one flow-through fraction (peak 1) and two eluted protein peaks (peaks 2 and 3), and different fractions were collected and dialyzed for 24 h against 20 mmol·L−1 phosphate buffer (pH 7.4). Compared to the control (AFB1 with 20 mmol·L−1 phosphate buffer (pH 7.4)), peak 2 (40–50 mL) was associated with the highest AFB1-degrading activity (3.85 × 103 U·mg−1) (3 U·mg−1), peak 2 (50–80 mL) (2.39 × 103 U·mg−1), peak 3 (80–95 mL) (1.73 × 103 U·mg−1) and peak 3 (95–100 mL) (≈ 0.00 U·mg−1). The specific activity after each step of purification is shown in To isolate the BADE responsible for AFB U·mg−1) , followe1 degradation by BADE  from isolate L7 varied as a function of pH , the degradation ratio of AFB1 in the presence of BADE increased significantly at pH 4.0 (10.40%), pH 5.0 (14.30%), and pH 7.0 (30.73%). The degradation ratio of AFB1 without BADE was 13.52% at pH 8.0  among different pH values, indicating that pH 8.0 is the optimal pH for BADE activity.The extent of AFBon of pH . There wshown in , and the1 degradation among the no-ion condition, Zn2+, Mn2+, Mg2+, Cu2+, and Li+ in the absence of BADE, and there was little degradation. Compared to the no-ion condition in the presence of BADE, BADE activity was inhibited by Zn2+, Mn2+, Mg2+, and Li+ (10 mmol·L−1) but was enhanced in the presence of Cu2+  activity was evaluated. There was no significant difference in AFB of Cu2+ .1-degrading activity of BADE  from isolate L7 varied as a function of temperature , the degradation ratio of AFB1 in the presence of BADE increased significantly at 16 °C (19.50%), 28 °C (24.40%), 37 °C (34.27%), 55 °C (27.01%), and 70 °C (47.51%). The degradation ratio of AFB1 treated with BADE increased following the increase in temperature, and the degradation rate at 70 °C was the highest (47.51%) among different temperatures, indicating that 70 °C is the optimal temperature for BADE activity.The AFBperature . There wB. shackletonii has AFB1-degrading activity. B. shackletonii strain LMG 18435T, a strictly aerobic bacterium isolated from the eastern lava flow of northern Candlemas Island in the South Sandwich archipelago, was reported to be capable of decomposing large molecules into organic acids [B. shackletonii strain K5 isolated from a biotrickling filter of a coal-fired power plant in Guangzhou, China showed a strong capacity for producing polyhydroxybutyrate [This is the first study reporting that ic acids . In addibutyrate .1 in solution could be effectively degraded by the culture supernatant of B. shackletonii L7. This was similar to previous findings demonstrating that extracellular extracts from R. erythropolis liquid cultures degraded AFB1, with only 33.2% residual toxin after 72 h [10 resting cells/mL of Fusarium aurantiacum for 4 h completely removed AFM1 (8 µg·mL−1) [R. erythropolis [S. maltophilia 35-3 [1 by the culture supernatant produced without pre-exposure to AFB1 showed the constitutive activity of B. shackletonii L7. The AFB1-degrading activity of the culture supernatant increased from 47.6% to 70.1% after ultrafiltration, and was decreased from 77.9% to 15.3% upon treatment with proteinase K and SDS, similar to the trends observed for the culture supernatants of R. erythropolis, M. fulvus ANSM068, and S. maltophilia [1-degrading enzyme from B. shackletonii is an extracellular enzyme, in contrast to intracellular AFB1-degrading enzymes isolated from fungi [We demonstrated that AFBter 72 h . Incubatµg·mL−1) . Similarhropolis  and S. mlia 35-3 , degradatophilia ,19,20. Tom fungi ,13.1 detoxification over time indicated that the AFB1-degrading enzyme from B. shackletonii was stable at 37 °C for one week. Similarly, a 66.8% reduction in AFB1 was observed over 72 h in the presence of R. erythropolis culture supernatant [1 level was reduced by 70%–80% within 36 h and AFB1 was completely degraded after 72 h by liquid cultures of Mycobacterium strain FA4 [1 was quickly removed from liquid by binding to lactic acid bacteria, although some of the toxin was released over a prolonged period of incubation [1 reduction, given the continuous increase in detoxification by strain L7 over time. To further verify the role of binding (or adsorption) in the observed reduction in aflatoxin concentration, bacterial pellets of B. shackletonii L7 cultured for 24 h were washed twice with and resuspended in phosphate buffer . The resuspended liquid (0.026 g/mL) was sterilized at 121 °C for 20 min and was used for the AFB1 degradation test, using phosphate buffer rather than the resuspended liquid as a control. AFB1 concentration did not decrease relative to the control upon treatment with the resuspended liquid; the AFB1 degradation rate was 0% (data not shown). This indicates that binding does not play a role in AFB1 reduction.The increase in AFBernatant , whereasrain FA4 . AFB1 wacubation ,19. Bind1-degrading enzymes were purified from the B. shackletonii culture supernatant by ion-exchange chromatography on a DEAE-sepharose column. AFB1-degrading enzymes have been purified from the fungi A. tabescens by ion-exchange chromatography and chromatofocusing chromatography [P. ostreatus by chromatography using DEAE-sepharose and phenyl sepharose columns [1-degrading activity (3.85 × 103 U·mg−1) was purified 9.55-fold from B. shackletonii cultures with a 39.92% yield, implying a low processing cost [Methods for purifying enzymes from microorganisms should be selected based on the activity and yield of the active component. In this study, crude AFBtography , and fro columns . In our ing cost  which, aing cost , makes iB. shackletonii BADE was active at a broad range of pH values (pH 4.0–8.0) and temperatures (16 °C–70 °C), which is advantageous for AF degradation in the digestive tracts of animals. Importantly, there was no decrease in enzymatic activity when the culture supernatant of strain L7 was subjected to heat treatment. This was in contrast with a previous study in which AFB1-degrading activity in the culture supernatant of S. maltophilia 35-3 was abolished by heat treatment [B. shackletonii BADE is heat-stable, similar to the enzyme in thermophilic B. shackletonii K5 [reatment . These rtonii K5 .1-degrading activity of B. shackletonii BADE was influenced by the presence of metal ions: the activity was enhanced by Cu2+ (10 mM) and inhibited by Zn2+, Mn2+, Mg2+, and Li+ (10 mM), in agreement with earlier reports [2+ as an activator. Cu2+ may participate in redox reactions in electron transport, transferring an oxygen atom to the AFB1 substrate; oxidized AFB1 would then be hydrolyzed into non-toxic products [2+ inhibited AFB1 degradation by BADE, which was similar to the one observed in F. aurantiacum [2+ may cause a conformational change in the enzyme that leads to its inactivation or decreases its affinity for AFB1 [The AFB reports ,31. We sproducts . In contantiacum . Zn2+ mafor AFB1 .1 degradation products with the SOS Chromotest. The genotoxicity of AFB1 was significantly reduced by treatment with proteins or enzymes in B. shackletonii L7 culture supernatant in the presence of S9. It was previously demonstrated that treatment of AFB1 with enzymes altered the double bond of the furofuran ring, with consequent changes in the fluorescence and mutagenicity of the molecule [1 detoxification; we also showed that the chemical properties of AFB1 degradation products differed from those of the parent molecule, although the detailed mechanism of AFB1 degradation by B. shackletonii BADE has yet to be described.We evaluated the genotoxicity of AFBmolecule . In thisB. shackletonii is the only AF-degrading enzyme purified from a bacterium. Enzyme purification is the limiting factor in the industrial-scale production of AFs-degrading enzymes. Challenges associated with obtaining BADE by B. shackletonii fermentation include contamination of bacterial cultures and low yields. Nonetheless, our results suggest that treatment of food and feed with recombinant B. shackletonii BADE can potentially eliminate mycotoxin contamination.To date, BADE from B. shackletonii strain L7, which could degrade AFB1, AFB2, and AFM1. A presumed extracellular BADE was purified from strain L7 showed the highest activity at 70 °C and pH 8.0 in the presence of 10 mmol·L−1 Cu2+, and was the main protein involved in the detoxification of AFB1. These findings indicate that BADE from B. shackletonii L7 is a promising new agent for AF biodegradation. The cloning and expression of the BADE-encoding gene are currently underway in our laboratory.We isolated 1, AFB2, AFG1, AFG2, and AFM1 were purchased from Sigma-Aldrich . Stock solutions (500 µg/L) were prepared by diluting the standard solutions with methanol. Coumarin medium (CM) was prepared as previously described [Standard solutions of AFBescribed . NB was 1 were isolated on CM medium [1-degrading activity.A total of 247 samples were collected from farm soils, maize, and rice in different provinces in China in June 2013. Bacterial strains with the ability to degrade AFBM medium ; selecte1 solution  was added to 0.4 mL of microbial culture to obtain a final concentration of 100 µg/L. The detoxification test was performed in the dark at 37 °C without shaking for 72 h; sterile NB containing AFB1 at a final concentration of 100 µg/L was used as a control.Isolates were cultured in NB for 12 h. For inoculation, a 12-h culture broth (1 mL) was transferred to NB (20 mL) and cultured at 37 °C with agitation for 24 h in a shaking incubator. AFBv/v), and the solution was passed through a sterile 0.22-µm pore size filter  and analyzed by high-performance liquid chromatography using a C18 column  and methanol:water  as the mobile phase at a flow rate of 1 mL/min. AFB1 was derived by a photochemical reactor  and detected with a fluorescence detector. Excitation and detection wavelengths were set at 360 and 440 nm, respectively. Percent AFB1 degradation was calculated using the formula (1 − AFB1 peak area in treatment/AFB1 peak area in control) × 100%. AFB2, AFG1, AFG2, and AFM1 degradations were analyzed in a similar manner.The reaction mixtures were extracted three times with chloroform according to a standard protocol . The chlBiochemical and physiological analyses of isolate L7 were performed following standard protocols . Genomic1 degradation by cell-free supernatant, bacterial cells, and intracellular cell extracts of strain L7 was evaluated as previously described [1 degradation, as described above. NB was used rather than the supernatant as a control.AFBescribed ,19,38. B1 degradation was evaluated as described above. Phosphate buffer was used rather than the bacterial cell suspensions as a control.Bacterial cell pellets were washed twice with phosphate buffer  and resuspended in the same buffer. AFBg for 10 min at 4 °C, and passed through a sterile 0.22-µm pore size filter. The AFB1 degradation test was performed as described above. Phosphate buffer was used rather than intracellular cell extracts as a control.Cell pellets resuspended in phosphate buffer  were lysed on ice using an ultrasonic cell disintegrator . Cell lysates were centrifuged at 12,000× 1 solution (500 µg/L) was combined with 0.4 mL of culture supernatant in a 10-mL tube, and the mixture was incubated in the dark at 37 °C without shaking. For incubation time studies, degradation was measured at 1, 6, 12, 24, 48, 72, 96, 120, and 168 h. The effect of heat treatment was assessed by immersing the culture supernatant in a boiling water bath for 10 min. The effect of proteinase K treatment with and without SDS was evaluated as previously described [1 degradation and protein concentration, an Amicon Ultra-15 centrifugal filter unit  with a cut-off molecular weight of 3 kDa was used for ultrafiltration of the culture supernatant to obtain different protein concentrations. NB alone processed in a similar manner and treated with AFB1 served as a control.The culture supernatant of strain L7 was prepared as described above. A 0.1-mL volume of AFBescribed . To dete1 degradation products was evaluated using SOS Chromotest Basic S9 Activation Enzymes v.64  according to the manufacturer’s instructions. Cell viability was monitored according to alkaline phosphatase activity at 420 nm using a microplate reader [1 solution combined with NB was used as a positive control, AFB1 solution combined with culture supernatant of isolate L7 was used as a degraded sample, and methanol added to the culture supernatant of isolate L7 served as a negative control. After incubation in the dark at 37 °C for 72 h (at which point the AFB1 degradation ratio was 77.9% ± 2.3%), 10 µL of the diluted solution was dispensed into the appropriate wells, and a range of sample concentrations  was tested using dimethyl sulphoxide as a diluent and reagent blank and 2-aminoanthracene serving as a positive control for S9.The genotoxicity of AFBe reader ,40. SampB. shackletonii cultures were prepared in a 1-L working volume of NB (pH 7.4). A 10-mL volume of fresh NB was inoculated with strain L7 at 37 °C, with agitation at 180 rpm for 12 h; 10 mL of the culture was then transferred to 1 L of the same medium. After cultivation at 37 °C with shaking at 180 rpm for 72 h, cells were harvested by centrifugation at 8000 rpm for 10 min at 4 °C. The culture supernatant was separated from the cell pellet and passed through a sterile 0.22-µm pore size filter. The supernatant of the 1-L culture was heated for 10 min and then subjected to ultrafiltration with a cut-off molecular weight of 3 kDa at 5000× g for 20 min in a refrigerated centrifuge to obtain a final volume of 20 mL, yielding a sample that was concentrated 50-fold. The concentrated sample was dialyzed against 20 mmol·L−1 Tris-HCl buffer (pH 8.5) for 24 h at 4 °C.Large-scale −1 Tris-HCl buffer (pH 8.5) and were eluted with an increasing linear gradient (0%–100%) of 1 mol·L−1 NaCl with 20 mmol·L−1 Tris-HCl buffer (pH 8.5) at a flow rate of 2 mL·min−1 until no protein was detected. The flow-through and eluted enzyme fractions were collected and dialyzed for 24 h against 20 mmol·L−1 phosphate buffer (pH 7.4). Each fraction was concentrated by ultrafiltration with a cut-off molecular weight of 3 kDa; the final volumes of each fraction were 20 mL for Peak 1 (0–20 mL), 10 mL for Peak 2 (40–50 mL), 30 mL for Peak 2 (50–80 mL), 15 mL for Peak 3 (80–95 mL), and 5 mL for Peak 3 (95–100 mL). Protein concentration was determined by the Bradford method using Coomassie Brilliant Blue, and AFB1-degrading activity was evaluated as described above. After each fraction was incubated with AFB1 for 72 h, residual AFB1 was detected by high-performance liquid chromatography. To assess the degradation of AFB1 with the proteins in different peaks, AFB1 with 20 mmol·L−1 phosphate buffer (pH 7.4) was used as a control. One unit (U) of enzyme activity was defined as the amount of enzyme required to decrease the amount of AFB1 by 1 ng in 72h at 37 °C. This purification step yielded the active fraction, which was tested for AFB1-degrading activity. The molecular mass of the active fraction was verified by SDS-PAGE.The dialyzed concentrated sample was subjected to ion-exchange chromatography on a DEAE-sepharose column . Proteins were packed in the column pre-equilibrated with 20 mmol·LB. shackletonii L7 was determined by SDS-PAGE based on the method of Laemmli [The molecular mass of the purified BADE from  Laemmli  on a 12%−1 citrate phosphate, and to 7.0 and 8.0 with 0.1 mol·L−1 sodium phosphate. The effect of temperature on BADE activity was determined at 16, 28, 37, 55, and 70 °C in 0.1 mol·L−1 sodium phosphate buffer (pH 7.0). A 0.1-mL volume of AFB1 solution (500 µg/L) was added to 0.4 mL of BADE solutions with different pH and at different temperatures, with a solution at pH 7.0 and 37 °C used as a control along with solutions without BADE under the same conditions. The assays were performed in the dark at 37 °C without shaking for 72 h.To determine the effects of pH on BADE activity, the initial pH value was adjusted to 4.0 and 5.0 with 0.1 mol·L2+ (ZnSO4), Mn2+ (MnCl2), Mg2+ (MgCl2), Cu2+ (CuSO4), and Li+ (LiCl) solutions at a final ion concentration of 10 mmol·L−1 were added to 0.1 mL of AFB1 (500 µg/L) combined with 0.4 mL of BADE solution (pH 7.0). BADE solution without any metal ions (no ion) was also used as a treatment. To assess the degradation of AFB1 in the presence of metal ions, solutions without BADE under the same conditions were used as controls. The assay was performed in the dark at 37 °C without shaking for 72 h.To determine the effect of different metal ions on BADE activity, ZnDifferences between groups were evaluated by analysis of variance with the general linear model procedure in a completely randomized single-factor design using SAS software . F tests were performed at the 0.05 level of probability. When a significant F value was detected, significant differences among the means were assessed with Duncan’s multiple range test.


Listeria phages P100 and A511 at temperatures simulating the preparation of ready-to-eat meats. The phage infectivity after heating to 71°C and holding for a minimum of 30 s, before eventually cooling to 4°C were examined. Higher temperatures of 75, 80, and 85°C were also tested to evaluate their effect on phages thermal-stability. This study found that despite minor differences in the amino acid sequences of their structural proteins, the two phages responded differently to high temperatures. P100 activity declined at least 10 log (PFU mL-1) with exposure to 71°C (30 s) and falling below the limit of detection (1 log PFU mL-1) while, A511 dropped from 108 to 105 PFU mL-1. Cooling resulted in partial reconstitution of P100 phage particles to 103 PFU mL-1. Exposure to 75°C (30 s) abolished A511 activity (8 log PFU mL-1) and both phages showed reconstitution during cooling phase after exposure to 75°C. P100 exhibited reconstitution after treatment at 80°C (30 s), conversely A511 showed no reconstitution activity. Heating P100 to 85°C abolished the reconstitution potential. Substantial differences were found in thermal-stability and reconstitution of the examined phages showing A511 to be more thermo-stable than P100, while P100 exhibited reconstitution during cooling after treatment at 80°C which was absent in A511. The differences in predicted melting temperatures of structural proteins of P100 and A511 were consistent with the observed differences in thermal stability and morphological changes observed with transmission electron microscopy.The study evaluated the thermal-stability of  Listeria monocytogenes continues to be a pathogen of concern in ready-to-eat (RTE) meat products causing listeriosis, a life threatening foodborne disease that primarily affects older adults, pregnant women, newborns, and adults with weakened immune systems. Due to high mortality rate of approximately 25–30%, the disease ranks among the most severe foodborne illnesses   . Antimicproducts .TM P100 and LMP-102 are approved by regulatory agencies as processing aids, for use in raw and RTE foods to combat L. monocytogenes contamination  phages which are the main cause of fermentation failure in the dairy industry . Buzrul for 15 s , thermalfor 15 s  demonstrListeria phages P100 and A511 at temperatures simulating the preparation of RTE meats, which included heating to 71°C and holding for a minimum of 30 s, before eventually cooling to 4°C  predictor algorithm  obtained from The National Microbiology Laboratory, Canadian Science Centre for Human and Animal Health  was used. Overnight cultures of L. monocytogenes were prepared by transferring a single colony of L. monocytogenes strain 08-5578 to 5.0 mL TSB  in a 15 mL screw cap, sterile Falcon tubes  in a rotary shaker and incubated for 18 h at 37°C and 120 rpm.Listeria phage P100 was obtained from MICREOS Food safety, Inc. (Netherlands) in PBS at concentrations of 1010–1011 PFU mL-1. Listeria phage A511 was obtained from The Félix d’Hérelle Reference Center for Bacterial Viruses, University of Laval . Phage A511 was propagated as described by L. monocytogenes subculture (109 CFU mL-1) and 100 μL of phage A511 (109 PFU mL-1) were added to 4 mL of top agar supplemented with CaCl2 . The solution was uniformly mixed and poured onto sterile TSA plates (Fisherbrand). Plates were incubated at 30°C for 18 h to form a top agar layer of phage-host co-culture. After incubation, 5 mL of SM buffer  was added to the plates to cover the surface of top agar entirely and refrigerated at 4°C overnight. After refrigeration all the liquids were extracted using a micropipette and filtered through 0.22 μm membranes. The filtrate was retained and stored at 4°C until use (ntil use . The tit10-11 PFU mL-1 and A511 at 109 PFU mL-1 (highest titre obtained by the propagation method indicated in the previous section) were used in the thermal stability experiment. An aliquot of 10 mL of P100 or A511 at 4°C was added to the heating solution and temperature monitored till the heating solution reached the respective treatment temperatures following which 1.0 mL sample was taken at t = 0 and then every 15 min for 75 min. Due to instability of phages in the 65°C treatment, samples were taken every 2.5 min for 20 min to assess thermal stability. Samples were immediately diluted in SM buffer and the phage titers determined with the phage overlay assay .The heating vessels used were standard 160 mL screw cap glass dilution bottles. Each bottle contained 90 mL of SM buffer (pH 7.5) with a magnetic stir bar. Prior to inoculation, SM buffer was equilibrated to the test temperature of 45, 55, or 65°C in a circulating water bath . In the case of all experiments described in this manuscript the treatment temperatures were maintained within ± 0.5°C. During heating, the water level was maintained at least 2.0 cm above the level of the treatment solution at all times and the magnetic stirrer operated using a MS-01 four position magnetic stirrer (Rose Scientific). Phage P100  at 10ay assay . All exp11 PFU mL-1) or A511 (109 PFU mL-1) at 4°C was added to the treatment solution and placed in a heated circulating water bath set at 72°C (temperature of the water bath was set to 1°C greater than the desired peak heating temperatures) and the temperature of the treatment solution monitored using a digital thermometer . Aliquot of 1.0 mL samples were taken at t = 0 (4°C), 40°C and then at every 10°C increase in sample temperature from 40 to 70°C, and 71°C (heating). The heating vessel was then held at 71°C for 30 s (holding) and another sample was taken. Immediately after heating and holding, the heating vessel was transferred to circulating cooling water bath, cooled using a glycol circulating cooling jacket set at -3°C. Aliquots of 1.0 mL samples were taken at every 10°C reduction in sample temperature, until the treatment solution temperature reached 4°C (cooling). Samples were immediately diluted in SM buffer and the phage titers determined with the phage overlay assay (5 and 108 (PFU mL-1) in treatment solutions were also examined at peak heating (71°C)-holding-cooling treatment and A511 with initial concentration of 105 PFU mL-1 in treatment solution was investigated at peak heating (75°C)-holding-cooling treatment. All experiments were performed in triplicate and each sample plated in duplicate.Treatments involved heating the phage suspensions from 4°C to 71, 75, 80, or 85°C, (referred to as heating) and holding for 30 s (referred to as holding) and then cooling back to 4°C (referred to as cooling) to check the stability of phages. The heating vessels used were standard 160 mL screw cap glass dilution bottles. Each bottle contained 90 mL of SM buffer (pH 7.5) with a magnetic stir bar. Prior to inoculation, the heating medium was equilibrated to 4°C. Then, 10 mL of P100  were conducted using the SAS Statistic Package , at a confidence interval of 95% to examine the variance in titre of the phage P100 and A511 at temperature points and between treatments. In cases where statistical differences between means were detected, Tukey’s test was applied for multiple pair-wise comparisons between treatment means.Listeria phages were examined at constant temperatures and heating-holding-cooling treatments. In order to estimate the thermal stability of P100 and A511 at constant temperatures, both phages underwent heat treatments of 45, 55, and 65°C. P100 and A511 are well characterized and their genes sequenced  in their tail interfaces. While highly similar at the primary sequence level, these phages exhibited different thermal responses. P100 and A511 were stable at 45°C for 75 min (data not shown). However, unlike A511, P100 exhibited thermal sensitivity at 55°C and lost ∼2 log PFU mL-1 activity. At the end of 20 min heat treatment at 65°C, A511 activity was reduced by 1 log cycle (PFU mL-1), while P100 was more sensitive with decreasing 4 log cycles in activity after 20 min (data are not shown). This is a significant (P < 0.05) decrease in D-value with the increase in treatment temperatures (Table 1). Increasing temperature to 65°C significantly (P < 0.05) decreased the D-values to 6.03 ± 0.47 and 16.80 ± 2.84 min for P100 and A511, respectively. Thermal stability of equenced . These pR2 was > 0.90 in all cases, indicating the data fits well with the linear regression model, except for A511 at 45°C (Table 1). At 45°C, A511 shows effectively no decline in titre, hence the near zero regression slope, and sensitivity to technical variation in measurements. Simple logarithmic inactivation of phages (Table 1). Although both phages infect the same host species, they show significant differences in their thermal-stability. It suggested that the major lethal event during heat treatments was due to the release of DNA from the phage particles  after 30 s of exposure to 71°C. When the same treatment was applied to A511, phage activity declined by only 3 log cycles (PFU mL-1), and retained activity even after 30 s of holding at 71°C, indicating that in spite of similar primary sequence of their structural proteins, A511 is more heat stable than P100. Release of phage DNA from viral capsids, as well as decomposition of the phages into head and tail structure and tail aggregation are known as common mechanisms observed for viruses thermal inactivation.In order to determine the thermal-stability of -1 of activity) after heating  followed by cooling to 4 ± 0.5°C . Hereafter, the term reconstitution refers to the phenomenon when phage infectivity/activity reaches below the limit of detection during heating and/or holding, while exhibiting renewed infectivity during cooling phase. Since A511 did not exhibit a loss of the complete activity (below limit of detection) at 71°C, thermal-stability was tested to 75°C, a temperature which renders A511 inactive , in order to examine if phage particles are capable of reconstitution. In addition, this approach enabled us to also examine if the phenomenon of reconstitution was observed in P100 at temperatures higher than 71°C. Raising the maximum temperature to 75°C yielded complete inactivation of P100 , while complete inactivation of A511 was observed after 30 s of holding . Cooling to 4.0°C after heating to 75°C resulted in partial reconstitution for both phages . This reconstitution activity was not observed when A511 was exposed to 80°C , yet P100 particles reconstituted after 30 s of exposure to 80°C . Despite the similarity of the two phages, A511 exhibited more thermo-stability compared to P100, but failed to show the same level of reconstitution. Raising the temperature to 85°C abolished the reconstitution ability for P100 . Considering the thermal inactivation and reconstitution of these phages, it could be speculated that although A511 had higher thermal stability during the heating stage compared to P100 , lack of reconstitution in A511 during the cooling stage compared to P100 suggests that heating of A511 results in an irreversible damage to the phage particle compared to P100 where the damage was reversible.Unlike previous studies examining the effect of heat on LAB and enterobacterial phages , P100 exTable 2 shows the temperature and phage population when reconstitution was observed, as well as phage population at the end of the heating-holding-cooling trial (4°C). There were an inverse relationship between peak heating temperature and the temperature at which reconstitution was observed. With an increase in peak heating temperature from 71 to 75 or 80°C, reconstitution was observed at 60, 50, and 20°C, respectively. The reconstitution of Listeria phages significantly (P < 0.05) decreased with an increase of temperature. P100 showed reduced reconstitution from 80°C (3.01 ± 0.36 log PFU mL-1) compared to lower peak temperature of 71°C (3.59 ± 0.04 log PFU mL-1). Similarly, A511 reconstitution of 2.84 ± 0.18 log PFU mL-1 was observed at 75°C, while no phage was detected after cooling following a peak heating temperature of 80°C (Table 2). In treatments with higher peak heating temperatures, cooling to lower temperatures was required to detect reconstitution (Table 2). It is possible that higher peak heating temperatures could result in irreversible damage/denaturation of the phage particles. This was evident from our results when phage suspension treated to higher peak temperatures, required cooling to lower temperatures to demonstrate the reconstitution phenomenon.Bacillus phage SPP1 in a buffer solution [500 mm NaCl and 50 mm Na2HPO4 (pH 8.0)] with a Fluorescence-based Thermal Shift Assay. They reported a loss of protein gp12 capsid fiber structure between 30 and 45°C and unfolding of polypeptide chains of gp12 was observed between 45 and 80°C. However, fast (<1 min) or progressive cooling of the sample back to 10°C led to complete reconstitution of the secondary, tertiary, and quaternary protein structures identical to that of the untreated proteins. Phenomenon of reconstitution for T4 phage tail particles previously was reported by Listeria phages relevant to food protection. To date, no reconstitution of Listeria phages undergoing heating process has been reported. According to these results, it is possible to conclude that the P100 and A511 proteins have characteristic to retain a native folded state when exposed to denaturing conditions to some extent of temperature elevation. It was more evident for tail structural proteins, especially for P100. The irreversible inactivation of phages occurred at temperatures >80°C due to irreversible unfolding and dissociation of structural proteins.Viral inactivation by thermal treatment has been previously studied , yet rec5 and 108 (PFU mL-1) were examined at heating 71°C-holding-cooling, phages could no longer be detected at 70°C , while phage particles were still detected (102-3 PFU mL-1) at 70°C during the same treatment when initial concentration of phage was 1010 PFU mL-1 . Reconstitution of P100 (108 PFU mL-1) was observed during cooling starting at 50°C , while the same treatment with initial concentration of 1010 PFU mL-1 resulted in reconstitution during cooling starting at 60°C . P100 with initial concentration of 105 PFU mL-1 exhibited no reconstitution of phage particles with the same treatment . When phage A511 with 105 PFU mL-1 were examined at heating 75°C-holding-cooling, no reconstitution activity were observed , while, partial reconstitution was observed with higher initial concentration of 108 PFU mL-1 . It is possible that the lower reconstitution ability and thermal stability observed in less concentrated phage solutions might be due to a dilution effect on phage population.Phages were tested with the same heating-holding-cooling protocol at different concentrations to evaluate the reconstitution activity. The lowest peak heating temperature at which complete inactivation and reconstitution was observed was picked to determine any correlation between phage concentrations and thermal-stability. Therefore, peak heating temperatures of 71°C and 75°C was used for P100 and A511, respectively. When P100 with initial concentrations of 10Figures 4A1,B1 show untreated controls of P100 and A511, respectively, which feature long, contractile tails and isometric capsids that are characteristic of Myoviridae family in the order Caudovirales  resulted in a mixed population of fully disrupted virions, aggregated tail . Similar observations were made in treatment to 75 and 80°C  with tail fibers and baseplates aggregation  showed only dark isometric capsid structure indicating that they were empty of DNA. TEM images of phage A511 from heating to 75°C displayed less tail aggregation with more visually intact phages  suggesting higher thermal stability which is in agreement with results presented in Table 1 and Figure 2. Samples treated to 80°C exhibited empty phage capsid without tails similar to that observed for P100 heated to 85°C .Transmission electron microscopy allowed us to visualize the physical effects of high temperature on P100 and A511. ovirales . HeatingFigure 4A resultedFigures 2).Transmission electron microscopy observations suggested that tail component of the phage were more thermosensitive and denatured at lower temperatures compared to capsid. Similar observation were made by In order to compare the differences between thermal-stability of P100 and A511 at the protein sequence level, relative estimate of minimum melting temperatures of structural proteins were determined using Tm predictor algorithm . Phage sTable 3. The P100 putative head-tail connector, predicted tail components such as tail fiber proteins, tail tip-collar, tail initiator, host recognition protein and baseplate hub, all were predicted to have lower melting temperatures than A511. The result is consistent with our observation of tail aggregation of P100 when exposed to heat. Portal proteins of A511 were predicted to melt at lower temperatures than P100 portal. Therefore, it is likely that heating A511 to 80°C caused the damage to the portal complex, disintegration of phage into head and tail and leakage of DNA (irreversible), while P100 retained the portal complex when exposed to 80°C (Table 3). This might be a plausible source of difference in ability of P100 and A511 to reconstitution after heating to 80°C and during cooling to 4°C.When comparing the primary sequence level between P100 and A511, 22 structural protein pairs were predicted as present in both phages. Minimum melting temperatures of these structural proteins were estimated using the Tm predictor and are presented in Listeria phages P100 and A511 at temperatures simulating the preparation of RTE meats. P100 and A511 are very similar but not identical , and they did not follow the same trend in thermal-stability and phage particle reconstitution, indicating thermal-stability is phage particle dependent. P100 showed more sensitivity to heat, being fully inactivated after exposure to 71°C and holding 30 s, yet was able to reconstitute even from heating to 80°C. However, phage A511 showed less sensitivity to heat and was not fully inactivated by exposure 71°C but didn’t show reconstitution after heating to 80°C. TEM visualization showed the detrimental effect of heat on phage structure causing tail aggregation, detachment of phage head and tail and generation of empty capsid at higher temperature. While there are many similarities between melting temperature of functional proteins in P100 and A511, there are specific differences as well, notably among structural proteins based on Tm prediction, which would explain the plausible source of difference in ability of P100 compared to A511 in reconstitution after heating to 80°C and during cooling to 4°C. Further analysis by nuclear magnetic resonance spectroscopy (NMR) or circular dichroism (CD) measurements for protein structure determination and folding and unfolding of proteins would help to understand these differences. Evaluation of the thermal stability of these phages in food matrix and their efficacy to control L. monocytogenes is required to determine if phages can be used as an additive in foods.The study evaluated the thermal-stability of HA performed all of the bacterial and phage assays presented, phage propagation, sampling, and data analysis. HA and DR both performed transmission electron microscopy and interpreted the resulting images. DR analyzed and interpreted the predicted protein melting temperature data. HA, DR, AK, SB, and L-TL contributed to experimental design for all assays presented. HA wrote the manuscript with input from SB, AK, DR, and L-TL. SB is the principle investigator of the project who was responsible for preparation of project proposal, procure funding, resource allocation and along with the AK and L-TL provided overall guidance and mentorship throughout the scope of this project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GW and handling Editor declared their shared affiliation.


Nucleic acid aptamers hold promise as therapeutic tools for specific, tailored inhibition of protein targets with several advantages when compared to small molecules or antibodies. Nuclear WW domain containing E3 ubiquitin ligase 1 (WWP1) ubiquitin ligase poly-ubiquitinates Runt-related transcription factor 2 (Runx2), a key transcription factor associated with osteoblast differentiation. Since WWP1 and an adapter known as Schnurri-3 are negative regulators of osteoblast function, the disruption of this complex has the potential to increase bone deposition for osteoporosis therapy. Here, we develop new DNA aptamers that bind and inhibit WWP1 then investigate efficacy in an osteoblastic cell culture. DNA aptamers were selected against three different truncations of the HECT domain of WWP1. Aptamers which bind specifically to a C-lobe HECT domain truncation were observed to enrich during the selection procedure. One particular DNA aptamer termed C3A was further evaluated for its ability to bind WWP1 and inhibit its ubiquitination activity. C3A showed a low µM binding affinity to WWP1 and was observed to be a non-competitive inhibitor of WWP1 HECT ubiquitin ligase activity. When SaOS-2 osteoblastic cells were treated with C3A, partial localization to the nucleus was observed. The C3A aptamer was also demonstrated to specifically promote extracellular mineralization in cell culture experiments. The C3A aptamer has potential for further development as a novel osteoporosis therapeutic strategy. Our results demonstrate that aptamer-mediated inhibition of protein ubiquitination can be a novel therapeutic strategy. E. coli, and inhibition of WWP1 to maintain Runx2 levels is a promising therapeutic strategy to stimulate osteoblast function. In mammalian bone, the osteoblast is exclusively responsible for the deposition of bone matrix in the form of hydroxyapatite crystals together with various structural proteins . The ost15) is exposed to a target, washed to exclude non-binding species, and eluted to recover binding species. Second, the pool is PCR amplified by having included 3’ and 5’ primer binding regions. Finally, the sequences are enriched by repeating the process successively while introducing increased stringency . The SELEX iterations are continued until enriched pools of homologous sequences are revealed, which then hold the potential to be tightly binding and specific [KD values in the µM to nM range with aptamer affinity maturation developed to increase affinity and specificity [Nucleic acid aptamers are short, single-stranded nucleic acid chains evolved to bind specifically to a given target. Riboswitches can be considered as natural non-coding RNA aptamers which regulate gene expression upon binding a metabolic target . Since 1specific ,11. Aptacificity . Aptamercificity ,14,15. Scificity ,17. Due cificity ,19, diagcificity ,21,22, acificity . Importacificity .Aptamers have traditionally focused on extracellular targets. However, polynucleotides such as aptamers can enter the intracellular space by either: (1) extraneous means such as liposomes, polymers, viruses, microinjection, electroporation, particle bombardment, calcium phosphate precipitation and ultrasound, or by (2) inherent mechanisms such as endocytosis or pinocytosis ,26,27,28Previously, our group selected aptamers which bind and inhibit the activity of the extracellular protein sclerostin, which is a known negative regulator of bone formation . Here, wE. coli, truncations of the smaller yet active HECT domain were used as the targets for aptamer selection. The HECT domain has two lobes: the N-lobe is involved in E2 binding and the C-lobe is important in ubiquitination [As it is not possible to express the full length WWP1 protein in tination . Rotatiotination , hence tAs shown in To begin, we used the entire HECT domain for the first 8 rounds of DNA aptamer selection. Important to its inhibition, HECT domain requires a flexible hinge loop, the binding of an E2 adapter, and a catalytic cysteine residue for activity A. We the#A when constant region is included and C#B without. The relationships between the variable regions is somewhat scattered—C3 is more closely related to C1 while C2 is completely unrelated. These three sequences were chosen to move forward in the project due to their higher copy number in sequencing data.We performed 4 further SELEX lobe-specific rounds (after the 8 round HECT domain pre-enrichment). Cloning and sequencing identified 31 aptamer sequences against the C-lobe truncation B, annotaTo compare the predicted secondary structures of the three aptamers, mFold software  was used . The strKD roughly in the low µM range was estimated for aptamer C3A, while other C-lobe aptamers and controls did not produce an inflection point with which to base a determination but with clearly weaker binding. Therefore, in later experiments we focused on aptamer C3A as the most promising of the three aptamer candidates.We next investigated the relative affinity of the DNA aptamers for the protein target relative to control. To demonstrate qualitative binding of the C-lobe aptamers to our recombinant HECT, Electrophoretic Mobility Shift Assay (EMSA) was employed . EMSA isThe HECT domain’s ability to self-ubiquitinate with the requirement of an E1 and E2 protein in vitro provides an approach to observe the kinetics of HECT domain activity. When the HECT domain is ubiquitinated, the HECT domain-ubiquitin conjugate is observed by SDS-PAGE appearing several kilodaltons above HECT and can be used for quantitation C. We usemaxv and MK are tabulated in 50 of approximately 100 µM. To determine mode of inhibition, two different C3A concentrations were tested (100 µM and 200 µM) while varying the concentration of ubiquitin substrate to determine MK and maxv as shown in vmax while the apparent MK remained similar. These results imply that the aptamer acts as a non-competitive inhibitor of ubiquitination.The resulting Fixed and stained SaOS-2 cells were imaged after the transfection process to investigate differences in localization. These stained images were used to compare aptamer C3A and control, both with and without transfection reagent as shown in After determining that aptamer enters the cells without transfection reagent, we compared localization over time by incubating aptamer C3Awith SaOS-2 cells without transfection reagent over 5 time points B. All apAptamer selections were begun with an entire HECT domain ‘pre-selection’. Subsequent selections against N-lobe, C-lobe and HECT pool showed enrichment of Aptamers only against C-lobe. This indicated that the C-lobe likely had selected tight-binding aptamers early in the selection process. We observed that it was necessary to retain the flanking constant regions in the C-lobe aptamers. Two classic examples of aptamers for which the PCR constant regions were not necessary for binding are pegaptanib and the anti-thrombin aptamer, but others have been reported where constant regions were required for binding ,38. We sKD of our C3A could be estimated in the low µM range. We were not able to obtain estimations from our other aptamers and control because they did not bind sufficiently at the tested range of concentrations, but one may conclude their binding was far weaker  guidelines from a human liver cDNA library. The following sequences were those for which the expression inserts were designed: HECT Domain (bp 1916–3047 NCBI Reference Sequence: NM_007013.3), C-lobe (bp 2687–3047 NCBI Reference Sequence: NM_007013.3), N-lobe (bp 2342–2587 in NCBI Reference Sequence: NM_007013.3). The resulting sequences, once inserted into pET28a(+)  vector, gave an N-terminal hexahistidine tags. Subcloning was initiated by 1% agarose gel purification of the PCR product with Platinum® Pfx Polymerase (Invitrogen) of each insert (approx 20 cycles) using QIAquick® gel extraction kit . Inserts were then digested with EcoRI and NdeI , 1% agarose gel purified and extracted, and the concentration of the insert and vector was then determined using absorbance at OD260/280 nm. Approximately 20 ng of insert and 10 ng of vector were put into ligation reaction with 1 µL ligase in total of 10 μL per instructions of T4 DNA ligase (New England Biolabs). A frozen eppendorf of XL-1 Blue E. coli  provided in house was made competent and transformed by roughly following the pET System manual (Novagen). Colonies were selected on 50 µg/mL kanamycin agar plates, grown in 1 mL cultures for exponential growth phase, DNA was purified by QIAprep (Qiagen), and sequenced by Tech Dragon Limited™, Hong Kong, China.HECT, C-lobe and N-lobe were amplified per PlatinumE. coli BL21(DE3) using the vendor transformation protocol (Novagen®). Overnight starter cultures of 30 µg/mL kanamycin containing LB media seeded 2–3 L cultures of the same media. These large cultures were grown at 37 °C under shaking at 200 rpm for approximately 6 h at 37 °C and induced to express with 500 µM IPTG, and shaken at room temperature for 4–5 h  and 4 °C overnight (HECT domain). Cell pellet was harvested by centrifugation at 4000 rpm, for 25 min, at 4 °C. After careful decanting, cell pellet was stored at −20 °C until purification. Column load was prepared by resuspending cell pellets in 20 mL of sonication buffer (Tris/500 mM NaCl/20 mM imidazole/0.1% (v/v) TritonX (pH 7.4)) for every 500 mL of culture and supplemented with Complete EDTA-free EASYpack protease inhibitor cocktail  followed by sonication for 10 min on ice at 30% amplitude. Cell lysate was centrifuged at 13,000 rpm for 25 min at 4 °C. Supernatant was filtered and loaded onto His-trap HP Ni2+ affinity column  using a peristaltic pump. After loading, the column was transferred to Vision™ Workstation . Real time UV trace was analyzed with the accompanying Vision™ Software . A gradient was run from 0% mobile phase A (50 mM Tris/500 mM NaCl/20 mM Imidazole (pH 7.4)) → 100% mobile phase B (50 mM Tris/500 mM NaCl/500 mM Imidazole (pH 7.4)) over 20 min at 3.5 mL/min and ~2 mL fractions were taken by hand throughout the gradient. Fractions were stored at −20 °C. Overall, the proteins were of correct size, were derived from the correct sequence, had purities of around 95% (excluding UbE2D2), and maintained a solubility of around 50% of the starting fraction pool after one week at 4 °C. All proteins expressed in high yield (concentrations of 2–4 mg/mL) and signal during imidazole gradients generally coincided with predicted isoelectric points.The pET28a(+) (Novagen) vectors containing in-frame and un-mutated insert for all three truncations of HECT and pET-15b (Novagen) containing UbE2D2  were transformed into 2O and contained a total of 4.2 × 1015 DNA molecules of 1.15 × 1018 possible sequences from a variable region of N = 30. Determination of the amount of protein target to be used was based on the Ni-NTA Agarose Beads Handbook  estimate that approximately 3 µg of protein bind to 10 µL of bead solution. The amount of target protein necessary to saturate 10 µL of bead solution was determined by exposing the beads to an increasing amount of protein, washing with selection buffer (50 mM Tris/0.05% Tween-20/0 → 1.0 M NaCl (pH 7.3)), eluting with 1.0 M imidazole in selection buffer, and visualizing on SDS-PAGE. After washing beads with protein buffer (50 mM Tris/400 mM NaCl/0.05% Tween-20 (pH 7.3)), appropriate amounts of protein were added and beads, and washed 3 times again with protein buffer. A 25 µL aliquot of library was then diluted with 200 µL of selection buffer and added to the beads, gently mixed, and set for 1 min. The beads were then washed with 1 mL of selection buffer 6–12 times depending on the round of selection. Protein bound to DNA was eluted with 50 µL elution buffer  and 5 µL was carried on to PCR amplification with biotinylated reverse primer for ~10 cycles. The resulting double stranded amplification product from each consecutive round was separated by washing 50 µL of Dynabeads M-280 Streptavidin magnetic beads  with 1 mL separation buffer (50 mM Tris/0.05% Tween-20 (pH 7.3)), binding the entire 50 µL PCR product and 900 µL separation buffer with the washed beads, washing three times with separation buffer, and eluting with 50 µL of 100 mM NaOH. DNA containing NaOH was diluted with 150 µL TBS for neutralization. Progression of the selection process was monitored by PCR and PAGE. Pools of double stranded DNA from final rounds of selections were blunt end cloned into vectors using Zero Blunt® TOPO® PCR Cloning Kit (Invitrogen) and transfection of XL-1 Blue E. coli  was performed as previously mentioned. 50 colonies from 50 µg/mL kanamycin selective plates were grown in 2.5 mL cultures for approximately 10 h, and said cultures were prepared for sequencing using the QIAprep® Spin Miniprep Kit . Sequencing of plasmids was performed by Tech Dragon Limited, Hong Kong, China.Basic methodologies and tools for aptamer selection were adapted from our labs previous methods. A random DNA library was obtained from Tech Dragon Limited, Hong Kong containing 6.9 nanomoles of single stranded DNA of the following design: Aptamers→ 3’-CTAATACGACTCACTATAGG(N30)AAGCTTTGCAGAGAGGATCCTT-5’, Primers→ 3’-GATTATGCTGAGTGATATCC, TTCGAAACGTCTCTCCTAGGAA-5’-Biotin. DNA was reconstituted in MilliQ H® Company which specializes in ubiquitin assays. Active HECT domain and the human E2 protein (UbE2D2) were expressed in E. coli, purified as described previously and were kept at 100 ng/µL and 160 ng/µL, respectively, at −20 °C in ~25% glycerol. His6-tagged human recombinant Ubiquitin and recombinant Human His6-Ubiquitin E1 Enzyme (UBE1) (Boston Biochem®) were kept at 5 µg/µL and 250 ng/µL, respectively, at −20 °C. The reaction was optimized by starting at 250 ng E1, 300 ng, HECT, 320 ng E2, and 5 µg Ub, and then systematically altering assay parameters such as reaction volume, time, reagent amounts, and temperature. Finally, the reaction contained 2.6 µM HECT, 45.5 nM UBE1, 507.8 nM UBE2D2, and varying from 300 nM to 4.8 µM Ubiquitin depending on the experiment. The total reaction volume was 25 µL, was run for 1 h at 37 °C in assay buffer (50 mM Tris/50 mM NaCl/5 mM MgCl2/5 mM KCl/25 mM DTT/5 mM ATP (pH 7.5)), and stopped by addition of 25 µL SDS-PAGE loading buffer (100 mM Tris/40% v/v glycerol/8% w/v SDS/5% w/v beta-mercaptoethanol/0.04% w/v bromophenol blue (pH 7.5)) with a final gel load of 20 µL. Identity of the reaction product band (HECT~Ub conjugate) was confirmed by MS/MS . Gels were stained with Coomassie or Silver depending on need for quantitation and analyzed with ImageJ  and Prism®  softwares.A HECT ubiquitination assay was devised based on the general guidelines of the Boston Biochemv/v) Glycerol/0.02% (w/v) bromophenol blue (pH 6.8)) were created. The mixture was set for 60 min at 4 °C while gel was pre-run in TAE buffer at 100 V for equilibration. Samples were then loaded and run on 12% PAGE Gels which were prepared using vendor Midi Protean Cell guidelines  at 100 V for approximately 3 h until tracking dye reached bottom of the gel. Gels were stained with 3.5 µL of SYBR Gold Nucleic Acid Gel Stain  per 50 mL of TAE buffer and imaged in the UV Transilluminator Imaging System  and analyzed with ImageJ and Prism®. 6.5 pmol of aptamer was mixed with a series of concentrations of HECT, set for 1 h at 4 °C, run on 10% PAGE for 1.5 h at 100 V in 4 °C with minimal glycerol for gel loading, and stained with SYBR gold™.Briefly, 15 µL samples containing 11.3 µM → 293.9 nM HECT domain, 55.6 nM aptamer, and 4 µL of EMSA sample buffer (50 mM Tris/10% (® number HTB-85®), a human osteosarcoma line, were obtained in house labeled at passage 5. Cells were thawed, seeded, passaged according to the general guidelines of the Cell Culture Basics guide  in a Class II bio safety cabinet with McCoy’s 5a Media  supplemented with Penicillin/Streptomycin and 15% FBS, and centrifugation was performed on bench top centrifuge. Cells were stored at a controlled environment of 37 °C with 5% CO2 and viewed with an Eclipse TS100  light microscope at 4× and 10× magnifications daily. Samples to be prepared for fixed cell imaging, western blot, apoptosis, bone mineralization, and alkaline phosphatase activity were initiated by growing cells to ~80% confluency on either 6, 24, or 96 well cell culture plates . Cells were transfected with 100 ng of aptamer and control for 4 h per Lipofectamine 2000 (Invitrogen) guidelines using McCoy’s 5a Media (Sigma-Aldrich) without Penicillin/Streptomycin or FBS or treated with aptamer and control without transfection reagent for the same amount of time. Cells were then replaced with normal supplemented media after transfection, which took place once (T0) for fixed cell imaging, western blot, and apoptosis assays and three times (T0 then every 3 days) for mineralization and alkaline phosphatase assays. The cells remained alive for the course of the experiments.SaOS-2 Cells  were prepared with sterile technique by placing cover slips into the wells with forceps in the BSC hood, rinsing with cold methanol, and rinsing 3× with ice cold sterile PBS. Cells were seeded 1:4 and grown to ~90% confluency and then treated with aptamer and controls, with and without Lipofectamine™ 2000. At each time point, wells were washed with ice cold PBS, gently fixed with ice cold methanol at −20 °C for one hour and washed with ice cold PBS. After damping dry, Acrytol® mounting media  was used to mount slides, which were then dried overnight in the dark. Images were taken on an BX51 Fluorescence Microscope  using brightfield and the appropriate filters for Cy3 (red) and Hoechst (blue) at 40×. MetaMorph®  batch processing software was used to analyze at least 85 cells per sample.Cover slip containing Costar®) were used at 1/500 and 1/80,000, respectively, while generally following the Western Blotting Beginners Guide . 1 µL of B-Actin  stock was included with each primary antibody incubation as a loading control. Blots were imaged with UV Transilluminator Imaging System .Cells were first washed with ice cold PBS, then 200 µL of ice cold RIPA buffer (150 mM sodium chloride/1.0% Triton X-100/0.5% sodium deoxycholate/0.1% SDS (sodium dodecyl sulfate)/50 mM Tris (pH 8.0)) supplemented with complete EDTA-free EASYpack protease inhibitor cocktail was added and cells, scraped and recovered by pipette. Lysates were agitated at 4 °C for 30 min by light shaking and centrifuged for 20 min at 12,000 rpm on a X-15R bench top centrifuge (Beckman Coulter) at 4 °C before recovering supernatant. In this case, Rabbit derived Anti-Runx2  and Goat derived Anti-Rabbit IgG (whole molecule) Peroxidase conjugate  guidelines, read at 450 nm on a 200 SpectraMax 340PC 38 (Molecular Devices) and analyzed with Soft Max Pro (Molecular Devices) and Prism® (GraphPad) Software.Apoptosis samples were prepared as described in the cell culture section and were performed according to HT TiterTACS2O, and PBS added before digital photographs were taken.Assays to detect the calcium of bone deposition generally followed the Osteoblast Differentiation and Mineralization Guide . First, cells washed with ice cold PBS, fixed with ice cold methanol overnight, stained with 20 g/mL Alizarin Red S for 45 min at room temperature, washed 5 times with diHWe successfully generated a DNA aptamer (C3A) which binds to the C-lobe of WWP1 with a binding affinity of 1.9 μM. This aptamer was demonstrated to inhibit the ubiquitination activity of WWP1 in a non-competitive manner. The aptamer could internalize into SaOS2 cells even in absence of transfection agent. The aptamer was shown to stimulate extracellular matrix deposition relative to controls. Future work can improve binding affinity by microarray maturation, employ bioinformatics methods to modify the aptamer for greater functionality  or gener