Single Step Detergent Assisted Extraction and Solubilization of the Recombinant Matrix Protein of Newcastle Disease Virus (NDV) and Development of ELISA for Detection of Anti-NDV Antibodies in Chickens

Zahra Naz1,2, Fouzia Ismat1, Muhammad Saleem2, Mazhar Iqbal1,

Aamir Shehzad1 and Moazur Rahman1,2*

1Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

2School of Biological Sciences, University of the Punjab, Quaid-i-Azam Campus, Lahore, Pakistan

ABSTRACT

The matrix (M) protein is the most abundant structural protein in Newcastle disease virus (NDV), the causative agent of Newcastle disease (ND) in chickens. Owing to its highly conserved nature among NDV strains and also due to its pivotal role in the viral life cycle, the M protein can be employed as a promising diagnostic antigen for reliable detection of NDV infection in chickens. In the present study, we have devised a strategy for extraction and solubilization of the NDV M protein from Escherichia coli in a single step using a non-ionic detergent, lauryl-dimethylamine oxide (LDAO), enabling the purification of the detergent-solubilized M protein in a soluble form through affinity chromatography without compromising the structural integrity of the protein. Using the purified M protein as a diagnostic antigen, an indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of anti-NDV antibodies in multiple serum samples collected from different poultry farms of district Faisalabad, Pakistan. The data presented here reveal that the recombinant detergent-solubilized M protein is an active, promising diagnostic antigen and can be exploited in an indirect ELISA for rapid and reliable detection of anti-NDV antibodies in chickens. Owing to the conserved nature of the M protein and the ease of extraction and solubilization in a single step using LDAO, the developed ELISA can be up-scaled for rapid and reliable detection of anti-NDV antibodies and/or NDV infection in a large number of serum samples collected from chickens originating from different sources (poultry farms).

Article Information

Received 13 September 2021

Revised 27 October 2021

Accepted 14 November 2021

Available online 27 January 2022

(early access)

Published 25 August 2022

Authors’ Contribution

Conceptualization and supervision: MR. Formal work and analysis: ZN, FI, MS, MI, AS and MR. Writing original draft: ZN. Reviewing and editing: FI, MS, MI, AS and MR. Funding acquisition: MR and MI.

Key words

Matrix protein, Newcastle disease virus, ELISA, Chicken, LDAO

DOI: https://dx.doi.org/10.17582/journal.pjz/20210913180900

* Corresponding author: [email protected], [email protected]

0030-9923/2022/0006-2765 $ 9.00/0

Copyright 2022 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Newcastle disease (ND), also known as Ranikhet disease, is a highly contagious and deadly viral disease of chickens which poses a constant threat to the poultry industry worldwide (Suarez et al., 2019). ND is caused by Newcastle disease virus (NDV) (Suarez et al., 2019), a member of the Paramyxoviridae family (Fernandes et al., 2014). Like other members of the Paramyxoviridae family, NDV harbors a lipid bilayer envelope which is derived from the membrane of the host cell that the virus infects to replicate (Lamb and Parks, 2007). NDV displays polymorphisms in morphology and the viral particle size (Suarez et al., 2019). NDV exists either in the form of a sphere, with a diameter ranging from 100 nm to 500 nm, or in the form of a filament which varies in length but typically has a width of 100 nm (Suarez et al., 2019). The virus is extremely stable over a wide range of pH and at sub-optimal temperatures but is less stable at elevated temperatures (above 56 °C) (Ganar et al., 2014).

The genome of NDV consists of a negative-sense, non-segmented and single-stranded RNA molecule of approximately 15.2 kb that encodes several non-structural and six structural proteins (Ganar et al., 2014). The structural proteins include matrix (M), hemagglutinin-neuraminidase (HN), nucleoprotein (NP), fusion (F), phosphoprotein (P), and large polymerase (L) proteins (Gaikwad et al., 2019) (Fig. 1). Among the viral proteins, the M protein is a highly conserved structural protein encoded by different NDV strains (Shtykova et al., 2019), and is composed of 364 amino acids with a molecular weight of 40 kDa (Lamb and Parks, 2007). The dimeric M protein forms a grid-like array on the inner surface of the NDV membrane and interacts with the cytoplasmic tails of HN and F proteins in host cells (Russell and Almeida, 1984). In the virion, the M protein is associated with the lipid membrane, surface glycoproteins and nucleocapsid proteins (Panshin et al., 1997). The M protein is implicated in viral assembly and budding from the host cell surface as well as in the maintenance of the spherical structure of virions (Ganar et al., 2014; Shtykova et al., 2019). However, it has been found that the M protein does not play a significant role in enhancing the virulence of NDV (Iram et al., 2014).

 

A rapid and reliable diagnosis of NDV is highly important to prevent sudden ND outbreaks and the spread of the disease. Furthermore, it is also important to distinguish NDV infections caused by velogenic (highly virulent) strains from vaccination or lentogenic (less virulent) strains (Khan et al., 2010). Multiple conventional methodologies such as pathogenicity testing, serological tests, polymerase chain reaction (PCR)-based tests, and the HN assay can be used to diagnose ND (Getabalew et al., 2019; Khan et al., 2010). A diagnostic strategy that involves observing clinical signs and subsequently confirming NDV infection by conventional methods is not feasible for the characterization of ND in a large number of field samples, as conventional diagnostic methods take 4 to 7 days to confirm and validate an NDV outbreak (Bello et al., 2018). So, there is an urgent need to develop rapid and reliable laboratory-based diagnostic tests for the detection of NDV infection in a large number of field samples.

The detection of NDV infection by measuring the titer of anti-NDV antibodies in serum samples of unimmunized chickens using rapid and sensitive diagnostic assays is an attractive approach. Based on the level of the antibody titer, this approach also allows for estimating the time of infection as well as differentiation of birds exposed to even a mild NDV attack at any stage of their life from vaccinated chickens (Marquardt et al., 1985). Various types of serological tests have been reported which use NDV or its parts as diagnostic antigen(s) to detect anti-NDV antibodies in serum samples from infected birds (Berinstein et al., 2005; Choi et al., 2013; Koch et al., 1998; Makkay et al., 1999). Among these, two highly preferred tests for analyzing antibody titers are: the hemagglutination inhibition (HI) assay and the enzyme-linked immunosorbent assay (ELISA) (Yune and Abdela, 2017). In general, HI assays are not feasible for serodiagnosis of field samples consisting of different serotypes co-circulating in the field (Bello et al., 2018; Long et al., 2004). On the other hand, most of the reported ELISA tests for NDV have been developed using either the whole NDV or less conserved proteins (such as F and HN) of NDV as diagnostic antigens (Bello et al., 2018). Importantly, indirect ELISA tests which are based on the whole NDV or less conserved NDV proteins are not suitable for analyzing the titer of anti-NDV antibodies in serum samples of birds collected from different sources, most likely due to variations in NDV strains. To address these shortcomings, the conserved M protein of NDV can be exploited as a reliable diagnostic antigen to detect anti-NDV antibodies in serum samples of poultry birds infected with different NDV strains, since the M protein plays an important role in virus assembly and the amino acid sequence of the M protein remains conserved among different strains of NDV (Ganar et al., 2014; Seal et al., 2000; Shtykova et al., 2019). In the present study, we have evaluated the performance of the detergent-solubilized M protein of NDV as a diagnostic antigen in an indirect ELISA for rapid and reliable detection of anti-NDV antibodies in serum samples of chickens collected from different poultry farms. We previously reported a multi-step method for obtaining the NDV M protein expressed in Escherichia coli that consisted of refolding the M protein from inclusion bodies using urea, solubilization of the M protein using a non-ionic detergent (lauryl-dimethylamine oxide (LDAO)), and affinity purification using elution buffer that contained Tween-20 instead of LDAO (Iram et al., 2014). In the present study, we demonstrate that extraction and solubilization of the NDV M protein from E. coli using LDAO in a single step not only enhances the solubility and the yield of the recombinant M protein of NDV but also renders the detergent-solubilized M protein suitable for use in an indirect ELISA as a reliable diagnostic antigen for the detection of anti-NDV antibodies in multiple serum samples of chickens. Due to the ease of extraction and solubilization of the M protein in a single step using LDAO, the ELISA described here can be up-scaled, allowing rapid and reliable detection of NDV infection and/ or anti-NDV antibodies in a large number of serum samples of chickens collected from the field.

MATERIALS AND METHODS

Ethical approval

This work has received approval from the ethics committee of National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, and the work was performed following the guidelines of the institutional ethics committee.

Expression of the M protein

A widely used strategy to enhance the expression of recombinant proteins in E. coli is the use of engineered bacterial strains (Jana and Deb, 2005; Sørensen and Mortensen, 2005). To get the maximum yield of the M protein, therefore, we expressed the pHis8-MBP-Matrix plasmid, available in Structural Biology Lab (NIBGE) (Iram et al., 2014), in different strains of E. coli in order to overcome the codon usage bias for expression of the viral protein in the prokaryotic host (Mädje et al., 2012; Rosano and Ceccarelli, 2009). For this, pHis8-MBP (the vector-only control) and the recombinant plasmid construct (pHis8-MBP-Matrix) were transformed into Rosetta 2(DE3) and BL21 (DE3) strains of E. coli. Transformed cells were selected on LB agar media (2% agar, 1% tryptone, 0.5% yeast extract, 1% NaCl and 1% glucose) supplemented with antibiotics (25 μg/mL chloramphenicol and 100 μg/mL ampicillin for the Rosetta 2(DE3) strain, and 100 μg/mL ampicillin for the BL21 (DE3) strain). A bunch of identical colonies containing the vector-only control and the recombinant plasmid construct were inoculated in 10 mL LB broth supplemented with appropriate antibiotics, in separate tubes, and were incubated overnight in an orbital shaker at 37 °C and 225 rpm. Then, 500 mL of expression media (LB broth supplemented with 0.2% glucose and relevant antibiotics) were inoculated with 2% of the overnight culture and incubated at 37 °C and 225 rpm until the OD600 reached 0.6. The culture was then placed on ice for 20 minutes. Expression of the recombinant M (His8-MBP-Matrix) protein was induced by addition of 0.25 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After induction for 8 h at 22°C and 225 rpm, cells were harvested by centrifugation at 4000× g at 4°C for 20 minutes. Expression of the recombinant M protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Extraction and solubilization of the recombinant M protein

As detergents such as LDAO can enhance the solubility and prevent aggregation of the M protein (Arachea et al., 2012; Columbus et al., 2006; Seddon et al., 2004), either urea or LDAO was used in the lysis buffer in the present study to refold and obtain the recombinant M protein in a soluble form. For this, pelleted cells from a 20 mL culture of the transformed E. coli Rosetta 2 (DE3) cells, containing the over-expressed recombinant M protein, were resuspended in pre-chilled lysis buffers (1 g wet cell biomass/7 mL buffer) either containing urea (20 mM Tris HCl, pH 7.9, 300 mM NaCl, 2 mM β-mercaptoethanol (β-ME), 8 M urea) or LDAO (20 mM Tris-HCl, pH 7.9, 300 mM NaCl and 2 mM β-ME supplemented with 1%, 5% or 10% LDAO (v/v)). The cell suspension was homogenized at high pressure, 20 kpsi, using a cell disruptor. The cell lysate was centrifuged at 12,000× g, 4 °C for 20 min to separate soluble and insoluble (inclusion bodies) fractions, and analyzed by SDS-PAGE. The urea-solubilized fraction was directly used for nickel-affinity purification under denaturing conditions. The concentration of LDAO in the LDAO-solubilized fraction was decreased from 5% (v/v) to 1% (v/v) using dilution buffer (20 mM Tris-HCl, pH 7.9, 300 mM NaCl, and 2 mM β-ME) before purification of the recombinant M protein through affinity chromatography.

Purification of the recombinant M protein

The LDAO-solubilized fraction was passed three times through the nickel-nitrilotriacetate (Ni-NTA) agarose affinity resin (QIAGEN), pre-equilibrated with buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole, 0.04% LDAO (v/v), and 2 mM β-ME). The resin was washed with 30x column volumes (CV) of wash buffer (20 mM Tris-HCl, pH 7.9, 150 mM NaCl, 25 mM imidazole, 0.04% LDAO, and 2 mM β-ME). The recombinant M protein was eluted with 5× CV of elution buffer (20 mM Tris-HCl, pH 7.9, 150 mM NaCl, 250 mM imidazole, 0.04% LDAO, and 2 mM β-ME). The purity of the protein was qualitatively analyzed through SDS-PAGE. Elution fractions containing the purified recombinant M protein were pooled and dialyzed against the dialysis buffer (20 mM Tris- HCl, pH 7.9, 150 mM NaCl, and 0.03% LDAO) in order to reduce the concentration of imidazole. The dialyzed protein was concentrated using a concentrating unit (Amicon Centrifugal Filter Unit, 30 kDa molecular weight cutoff), and glycerol (50% final concentration) was added to the concentrated protein. The protein was stored at -20 °C until further use.

Development of a recombinant M protein-based ELISA

An indirect ELISA was developed using the recombinant M protein as an antigen. The antigen (200 µL; 4 µg/well) was used to coat the wells of a micro-titer plate, and the plate was incubated overnight at 4°C. Wells were then washed three times with phosphate buffer saline-Tween 20 (PBS-T) supplemented with 0.04% LDAO (200 µL/well). Non-specific binding to the surface can be minimized by saturating these unoccupied binding sites with a blocking reagent in order to prevent non-specific binding, 200 µL of the blocking buffer (5% BSA in PBS) was added to each well, and the plate was incubated at room temperature for 60 min. Wells were then washed as above and dried. Serum samples collected from naturally infected birds, showing clinical signs and symptoms of ND, from various poultry farms (namely, Rana Amjad Ali poultry farm, AAA poultry farm, NM poultry farm, SF poultry farm, NIAB), in district Faisalabad, Pakistan, were diluted (i.e., 1:50, 1:100, 1:200, 1:400) in PBS-T, and applied to the wells (100 µL/well). After incubation at room temperature for 2 h, wells were washed three times with PBS-T containing 0.04% LDAO (200 µL/well) and dried. The anti-chicken IgY (IgG) (whole molecule), alkaline phosphatase antibody produced in rabbits (A9171 Sigma) was diluted (1:25000) in PBS-T and added to each well (200 µL/well). After incubation at room temperature for 2 h, wells were washed three times with PBS-T supplemented with 0.04% LDAO (200 µL/well) and dried. To develop the signal, 100 µL of the alkaline phosphatase substrate (p-nitrophenyl phosphate disodium salt hexahydrate), freshly prepared in 0.1 M glycine buffer (pH 10.4) (0.1 M glycine, 1 mM MgCl2 and 1 mM ZnCl2), was added to each well. The plate was incubated at room temperature for 30 minutes, and observed for the development of the yellow colored product (para-nitrophenylphosphate (pNPP)). The reaction was stopped by addition of 3 M NaOH (25 µL/well). To analyze the antibody titer, the absorbance was measured at 405 nm using a microplate reader (Dia 710- Microplate Reader).

RESULTS

Expression analysis of the recombinant M Protein in different E. coli strains

To obtain the M protein in sufficient quantities, the protein was expressed in fusion with a C-terminal His8-MBP tag in Rosetta 2(DE3) and BL21 (DE3) strains of E. coli. In both the cases, the analysis of the protein expression by SDS-PAGE revealed the appearance of a dense band at ~83 kDa. However, the expression level of the recombinant M protein was higher in E. coli Rosetta 2(DE3) cells than in E. coli BL21 (DE3) cells. Furthermore, the expression of the recombinant M protein (His8-MBP-Matrix) in a soluble form increased by lowering the post-induction temperature of the culture to 22 °C, as analyzed through SDS-PAGE (Fig. 2).

 

Extraction and solubilization of the recombinant M protein

In our previous studies, the purification yield of the refolded M protein was too low (Iram et al., 2014). Therefore, E. coli Rosetta 2(DE3) cells containing the recombinant M protein were first lysed in the presence of 8 M urea (see Materials and Methods) in an attempt to improve the purification yield of the M protein. The soluble cellular content (supernatant) was applied to a Ni-NTA column, and the bound protein was eluted using elution buffer containing 250 mM imidazole and 8 M urea. However, the SDS-PAGE analysis revealed that the recombinant M protein could not be purified in the presence of 8 M urea (data not shown). To overcome the issue and obtain the protein in a soluble form, cell lysis and protein purification steps were performed in the presence of a non-ionic detergent, LDAO. For this purpose, different concentrations (1%, 5% and 10%) of LDAO were used in the cell lysate to enable the extraction and solubilization of the target protein. To this end, E. coli Rosetta 2(DE3) cells containing the overexpressed M protein were lysed in the presence of 1%, 5% and 10% LDAO in dilution buffer (20 mM Tris-HCl, pH 7.9, 300 mM NaCl, and 2 mM β-ME). The SDS-PAGE analysis revealed that the recombinant M protein was equally distributed in soluble and insoluble fractions obtained from cells lysed in the presence of 1% LDAO. On the other hand, the protein was predominantly extracted in the soluble form from the cell lysate treated with 5% LDAO (Fig. 3A, B). The SDS-PAGE analysis of samples from cell lysis with 10% LDAO showed diffused bands, forming streaks on the gel which may be attributed to a high concentration of LDAO (Fig. 3C).

 

 

Purification of the recombinant M protein

Based on the protein solubilization results, 5% LDAO was selected as the optimal concentration for solubilization of the recombinant M protein from lysed cells. The diluted supernatant (containing 1% LDAO, final concentration) was allowed to pass through the Ni-NTA resin and the bound recombinant protein was eluted with elution buffer containing 250 mM imidazole. Elution fractions were analyzed through SDS-PAGE, and the results showed that the recombinant M protein was purified in a stable form, with reasonable purity (Fig. 4), and in quantities sufficient for conducting serological studies.

Development of the M protein-based ELISA

An indirect ELISA was developed using the purified M protein as the coating antigen. Antisera samples collected from NDV-infected birds were used in 1:50 dilution for measuring the antibody titer against NDV (whole virus). Our results confirmed the presence of anti-NDV antibodies in all of the collected samples (Fig. 5), suggesting that the developed ELISA can reliably detect anti-NDV antibodies in serum samples of NDV-infected birds from different sources (poultry farms). Notably, the antibody titer in the serum samples from the Rana Amjad (layer) farm (serum samples 1 and 2) showed that a significant amount of the anti-NDV antibodies was produced in the chickens. Moreover, anti-NDV antibodies were confirmed in the serum samples of the chickens obtained from AAA farms (serum samples 3, 4) and NIAB (serum sample 7) but the antibody titer was less than that of the Rana Amjad (layer) farm. Antiserum samples from NM (serum sample 5) and SF farms (serum sample 6) were also confirmed for the presence of NDV-specific antibodies. None of the ELISA controls gave any significant signal at 405 nm. Antibody titers along with their standard deviations for the tested samples are shown in Figure 5. Based on these results, it can be concluded that the conserved M protein, which is detergent (LDAO)-solubilized, can be used as a reliable diagnostic antigen for the detection of anti-NDV antibodies in serum samples of chickens collected from different poultry farms.

Discussion

Every year, infectious diseases of chickens adversely affect the growth of the poultry industry across the globe. ND, caused by NDV, is a highly devastating disease confronting the poultry industry (Suarez et al., 2019). One of the approaches for mitigating the economic losses due to ND is the timely diagnosis of the disease in chickens so that preventive measures to control the spread of the disease to uninfected birds can be undertaken timely. A highly preferred technique for the detection of NDV or anti-NDV antibodies in antisera of infected birds is ELISA due to its reliability, high reproducibility, sensitivity and specificity (Makkay et al., 1999). To date, ELISAs based on various proteins of NDV have been developed for the detection of anti-NDV antibodies and/or NDV in infected birds. These ELISAs are either based on the structural proteins of NDV or the whole NDV (Gaikwad et al., 2019; Zhao et al., 2018).

 

Two structural proteins (NP and P) of NDV have been extensively exploited for ELISA development (Zhao et al., 2018). The NP protein is the most abundant viral protein expressed in infected cells, whereas the P protein is associated with the nucleocapsid in the virion (Ahmad-Raus et al., 2009; Errington and Emmerson, 1997; Kho et al., 2004; Panshin et al., 2000; Zhao et al., 2018). However, the variability and serotype specificity of both the proteins limit the use of the N or P-based ELISAs for the detection of different NDV serotypes (Zhao et al., 2018). Recently, structural proteins other than NP and P (such as HN and F) of NDV have also been used for the development of ELISAs. An NDV genotype 2.VII-specific concanavalin A (conA)-ELISA has been developed by targeting the HN protein (Moharam et al., 2021). For this purpose, the HN protein obtained through biophysical enrichment from purified NDV was used as an antigen (Moharam et al., 2021). However, the genotype specificity of the HN protein limits the use of the developed ELISA for the detection of NDV in samples from different sources. Based on the purified F protein as an antigen, an indirect ELISA has been reported for the detection of NDV in poultry (Astuti et al., 2020). Serum samples were collected from birds vaccinated with either the purified recombinant F protein or a commercially available vaccine (Astuti et al., 2020). Results of the study confirmed that the recombinant F protein can be used as an antigen for the detection of a specific immune response against the F gene/protein-based NDV vaccines (Astuti et al., 2020). However, it remains to be seen if the developed F protein-based ELISA can be used for the detection of NDV infection in unimmunized birds.

Indirect ELISAs which are based on the whole NDV have also been reported for the detection of anti-NDV antibodies (Häuslaigner et al., 2009; Oliveira et al., 2013). For instance, an indirect ELISA which is based on the LaSota strain of NDV has been used to detect antibodies against NDV in the antisera collected from immunized domestic geese and ducks (Häuslaigner et al., 2009). However, a major factor which limits the widespread use of indirect ELISAs is that these tests are not suitable for analyzing multiple antisera of birds which originate from different sources such as antisera collected from birds of different poultry farms, most likely due to variations in NDV strains.

In the present study, we have targeted the M protein of NDV for ELISA development due to a high degree of conservation of the M protein among different NDV strains. For this purpose, the recombinant M protein was extracted and solubilized in a single step using a non-ionic detergent, LDAO, and purified in large amounts in a soluble form using affinity chromatography. The purified M protein was used as a coating antigen to develop indirect ELISA as a rapid diagnostic test for NDV. Our results show that the anti-NDV antibody titers in the serum samples of infected birds collected from different poultry farms are significantly higher than that of uninfected (control) birds (Fig. 5), confirming that the detergent-solubilized M protein is a promising diagnostic antigen for the detection of anti-NDV antibodies and/or NDV in infected chickens from different sources. It is worth-noting that we previously documented a multi-step procedure for obtaining the NDV M protein from E. coli that involved refolding the M protein from inclusion bodies using urea, solubilization of the M protein using LDAO, and affinity purification using an elution buffer that contained Tween-20 instead of LDAO (Iram et al., 2014). In the current study, we have devised a strategy that allows extraction and solubilization of the recombinant M protein in a single step using LDAO, enabling the affinity-purified M protein to be used as a diagnostic antigen in an indirect ELISA for reliable detection of anti-NDV antibodies in serum samples of birds collected from different poultry farms. The single-step extraction and solubilization of the recombinant M protein using LDAO reduces the cost and time needed for obtaining the same amount of the protein through the multi-step procedure, thus opening up the possibility for up-scaling the developed ELISA presented in the current study for analyzing a large number of serum samples of NDV-infected birds from different poultry farms.

CONCLUSIONS

In summary, the recombinant M protein of NDV was successfully extracted and solubilized using LDAO in a single step and purified in large amounts in a soluble form through affinity chromatography. Considering the conserved nature of the M protein and an increased yield of the protein obtained without compromising its structural integrity, an indirect ELISA was developed using the LDAO-solubilized M protein as a diagnostic antigen. We demonstrate that the purified M protein can be used for the development of a cost-effective, rapid, and reliable ELISA for the detection of anti-NDV antibodies and/or NDV in serum samples of poultry birds collected from different sources.

ACKNOWLEDGEMENTS

This work was supported by the Higher Education Commission of Pakistan (grant No. 20-2138 (awarded to M. Rahman)).

Statement of conflict of interest

The authors have declared no conflict of interest.

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